Tissue-engineered intervertebral disc with living cells

ABSTRACT

The present invention relates to a tissue-engineered intervertebral disc (IVD) comprising a nucleus pulposus structure comprising a first population of living cells and an annulus fibrosis structure surrounding and in contact with the nucleus pulposus structure, the annulus fibrosis structure comprising a second population of living cells and collagen.

This application is a continuation of U.S. patent application Ser. No.16/442,535, filed Jun. 16, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/497,406, filed Apr. 26, 2017, now U.S. Pat. No.10,363,341, issued Jul. 30, 2019, which is a continuation of U.S. patentapplication Ser. No. 14/710,330, filed May 12, 2015, now U.S. Pat. No.9,662,420, issued May 30, 2017, which is a division of U.S. patentapplication Ser. No. 13/319,025, filed Dec. 10, 2012, now U.S. Pat. No.9,044,335, issued Jun. 2, 2015, which is a national stage applicationunder 35 U.S.C. § 371 from PCT Application No. PCT/US2010/033752, filedMay 5, 2010, which claims the priority benefit of U.S. ProvisionalPatent Application Ser. No. 61/175,680, filed May 5, 2009, which arehereby incorporated by reference in their entirety.

This invention was made with Government support under Grant Number DMR0520404 awarded by NSF. The United States Government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention is directed to tissue-engineered intervertebraldiscs and methods of their fabrication.

BACKGROUND OF THE INVENTION

Degenerative disc disease (“DDD”) is a leading cause of disability inthe U.S. and Europe, affecting an estimated 50 million people. As withother musculoskeletal ailments, DDD is a problem that grows as thepopulation ages. Medical options for treatment are limited to analgesicand anti-inflammatory drugs, which do not address underlying causes ofthe disease. Surgical options for the treatment of DDD are limited tofusion of spinal motion segments and implantation of motion-preservingdevices that have limited lifetimes due to material failure andbiocompatibility issues. An alternative to these approaches involvesregenerating the intervertebral disc (“IVD”) using tissue engineeringtechnology. IVD regeneration is particularly challenging, since IVDcontains two distinct types of tissue, the annulus fibrosus and nucleuspulposus, that are both important to the mechanical function of thedisc.

IVD degeneration is a leading cause of disability in the developedworld, with approximately 50% of the population over 50 years oldexperiencing prolonged back pain (Diwan et al. “Current Concepts inIntervertebral Disc Restoration,” Orthop. Clin. North Am. 3:453-64(2000)). Current medical treatment of disc degeneration is purelypalliative, focusing on relieving pain, but not restoring function. Theprimary surgical option for such patients, spinal fusion, altersmechanical loading of remaining IVDs, with pain and adjacent discdegeneration often following (Huang et al, “The Current Status of LumbarTotal Disc Replacement,” Orthop. Clin. North Am. 35:33-42 (2004)).

As such, significant attention has been paid to developing spinal fusionalternatives, including IVD replacement implants to enable motionbetween vertebral bodies. Such implants have begun to be used widely inclinical trials and practice including Acroflex (DePuy Acromed), SBCharite III (DePuy Spine), Maverick (Medtronic), and PRODISC® (SpineSolutions, Inc.). While these implants differ in specific design, allshare similar components, a combination of metal and plastic parts ofsimilar composition to those used in traditional hip and kneereplacements. Although promising, these implants are subject to thefailure modes experienced by other synthetic polymer/metal implantsincluding wear, fatigue, and loosening via osteolysis. In fact, thesecomplications are likely to be of even greater concern, given that theenvironment of the spine may not be able to clear wear debris asefficiently as synovial joints.

An alternative approach to restoring the function of spinal motionsegments involves developing biologically based implants using tissueengineering approaches. A properly tissue-engineered IVD tissue implantwould restore function and have the ability to continuously remodel in away similar to native IVD to enable long term function. The greatpromise of such an approach is tempered by the complexity of the task ofcreating a tissue with multiple components and complex organization.Most recent efforts in IVD tissue engineering have focused onregenerating individual components of tissue, the annulus fibrosus(“AF”) and the nucleus pulposus (“NP”).

There have been remarkably few studies examining the function ofengineered IVD tissues. The function of the IVD is mechanical, servingto maintain vertebral spacing and enable motion between vertebralsegments. Several in vivo studies have demonstrated the ability of celltransplantation techniques to maintain disc height (Meisel et al.,“Clinical Experience in Cell-Based Therapeutics: Disc ChondrocyteTransplantation A Treatment for Degenerated or Damaged IntervertebralDisc,” Biomol. Eng. 24(1):5-21 (2007); Sakai et al., “Immortalization ofHuman Nucleus Pulposus Cells by a Recombinant SV40 Adenovirus Vector:Establishment of a Novel Cell Line for the Study of Human NucleusPulposus Cells,” Spine 29(14):1515-23 (2004)). For in vitro studies,disc height is not an appropriate measure. Therefore, the best indicesof function are the mechanical properties of the tissue, such as thecompressive modulus, which is likely related to the ability to maintainheight when loaded. Despite the necessity of characterizing suchmechanical behavior, only three studies have measured mechanicalproperties of engineered IVD (Baer et al., “Collagen Gene Expression andMechanical Properties of Intervertebral Disc Cell-Alginate Cultures,” J.Orthop. Res. 19(1):2-10 (2001); Seguin et al., “Tissue EngineeredNucleus Pulposus Tissue Formed on a Porous Calcium PolyphosphateSubstrate,” Spine 29(12):1299-306 (2004); Mizuno et al., “Biomechanicaland Biochemical Characterization of Composite Tissue-EngineeredIntervertebral Discs,” Biomaterials 27(3):362-70 (2006)) with the mostrecent two demonstrating progression of mechanical function with time.Thus, while many cell types and scaffold materials have beeninvestigated, there is no consensus on a method to produce tissuesuitable for IVD replacement.

The native IVD has the capacity to regenerate and surgical strategieshave been explored that promote healing and repair of an AF defect.Regenerative strategies can be divided into cell therapy, gene therapy,and tissue engineering with scaffolds (Bron et al., “Repair,Regenerative and Supportive Therapies of the Annulus Fibrosus:Achievements and Challenges,” Eur. Spine J. 18(3):301-13 (2009);Hegewald et al., “Regenerative Treatment Strategies in Spinal Surgery,”Front. Biosci. 13:1507-1525 (2008)). Implantation of cultured autologousNP and AF cells into the intervertebral disc of animals has been used asan approach for potential future treatment of degenerative disc disease(Okuma et al., “Reinsertion of Stimulated Nucleus Pulposus Cells RetardsIntervertebral Disc Degeneration: An In Vitro and In Vivo ExperimentalStudy,” J. Orthop. Res. 18(6):988-97 (2000); Gruber et al., “The SandRat Model for Disc Degeneration: Radiologic Characterization ofAge-Related Changes: Cross-Sectional and Prospective Analyses,” Spine27:230-4 (2002)). Others have reported interim results of a humanrandomized trial using autologous NP cells derived from therapeuticdiscectomy that were cultured and delivered 12 weeks followingdiscectomy in patients with chronic back pain (Meisel et al., “ClinicalExperience in Cell-Based Therapeutics: Disc Chondrocyte TransplantationA Treatment for Degenerated or Damaged Intervertebral Disc,” Biomol.Eng. 24(1):5-21 (2007)). Their data suggests MR imaging improvementconsistent with increased proteoglycan matrix within the NP and areduction in low back pain at 2 years when compared to controls. Itwould be unlikely, however, that injection of cells would be effectivein cases of more severe disc degeneration. Attempts to use AF cells forregeneration are currently limited due to the problems encountered withisolation and proliferation of these cells in vitro (Bron et al.,“Repair, Regenerative and Supportive Therapies of the Annulus Fibrosus:Achievements and Challenges,” Eur. Spine J. 18(3):301-13 (2009)).

Aligned collagen fibril architectures have been generated by contractingcollagen gels under a variety of boundary conditions (Barocas et al.,“Engineered Alignment in Media Equivalents: Magnetic Prealignment andMandrel Compaction,” J. Biomech. Eng. 120:660-6 (1998); Grinnell andLamke, “Reorganization of Hydrated Collagen Lattices by Human SkinFibroblasts,” J. Cell Sci. 66:51-63 (1984); Thomopoulos et al., “TheDevelopment of Structural and Mechanical Anisotropy in FibroblastPopulated Collagen Gels,” J. Biomech. Eng. 127:742-50 (2005)). One groupused this technique to create circumferentially aligned fibrils byimposing an annular outer boundary on contracting collagen gels seededwith human-dermal fibroblasts (Costa et al., “Creating Alignment andAnisotropy in Engineered Heart Tissue: Role of Boundary Conditions in aModel Three-Dimensional Culture System,” Tissue Eng. 9:567-77 (2003)).The use of an inner mandrel has also been shown to produce alignedstructures in tissue-engineered blood vessels (Stegemann et al.,“Genetic Modification of Smooth Muscle Cells to Control Phenotype andFunction in Vascular Tissue Engineering,” Tissue Eng. 10:189-99 (2004)).However, none of these methods have been successfully applied to yield acomposite IVD with aligned collagen fibrils and AF cells around anengineered NP.

There remains a grand challenge to restore function to the spine byrepairing or regenerating IVD tissue. Such an approach is tempered bythe complexity of the task of regenerating a tissue with complexorganization. The native IVD functions via an intricate load sharingmechanism between its two principle components, the AF and the NP. It isthe complex architecture that is responsible for providing mobility tothe spine while handling the hoop, torsional, and bending stressesimposed upon it during motion of the spine. However, it is this samecomplexity that has left a need for an effective treatment fordegenerative disc disease.

The present invention overcomes these and other deficiencies in the art.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a tissue-engineeredintervertebral disc suitable for total disc replacement in a mammal. TheIVD comprises a nucleus pulposus structure comprising a first populationof living cells that secrete a hydrophilic protein and an annulusfibrosis structure surrounding and in contact with the nucleus pulposusstructure, the annulus fibrosis structure comprising a second populationof living cells and type I collagen. The collagen fibrils in the annulusfibrosis structure are circumferentially aligned around the nucleuspulposus region due to cell-mediated contraction in the annulus fibrosisstructure.

Another aspect of the present invention relates to a method offabricating a tissue-engineered intervertebral disc suitable for totaldisc replacement in a mammal. This method involves providing a first gelcomprising a first population of living cells that secrete a hydrophilicprotein; forming the first gel into a predetermined shape and size;providing a second gel comprising a second population of living cellsand type I collagen; contacting the formed first gel with the second gelat a region that extends circumferentially around the first gel; andstoring the first and second gels under conditions effective for thecollagen in the second gel to align circumferentially around the firstgel by self-assembly of collagen due to cell-mediated gel contraction inthe second gel. The first gel forms a nucleus pulposus structure and thesecond gel forms an annulus fibrosus structure surrounding and incontact with the nucleus pulposus structure, thereby fabricating atissue-engineered IVD suitable for total disc replacement in a mammal.

Yet another aspect of the present invention relates to a method offabricating a tissue-engineered intervertebral disc suitable for totaldisc replacement in a mammal. This method involves providing a first gelcomprising a first population of living cells that secrete a hydrophilicprotein; providing a second gel comprising a second population of livingcells and type I collagen; forming the second gel around a centralmandrel structure; storing the second gel under conditions effective forthe collagen in the second gel to align circumferentially around thecentral mandrel by self-assembly of collagen due to cell-mediated gelcontraction in the second gel; and replacing the central mandrel withthe first gel. The first gel forms a nucleus pulposus structure and thesecond gel forms an annulus fibrosus structure surrounding and incontact with the nucleus pulposus structure, thereby fabricating atissue-engineered IVD suitable for total disc replacement in a mammal.

The present invention provides new techniques to generate composite IVDtissue with an AF structure that more closely mimics the native IVD.

The present invention provides for circumferential fibril alignment intissue-engineered AF using cell-induced contraction of collagen gelsaround inner mandrels or nucleus pulposus structures. The directedcollagen gel contraction is used to generate circumferentially alignedcollagen in the AF portion of the composite implants. Further,additional methods of the present invention relate to an AF composed ofconcentric lamellae of aligned collagen that are like that of (or mimic)native tissue. These composite tissue-engineered IVD of the presentinvention with aligned collagen structure have demonstrated remarkableability to restore function in rats that have undergone lumbar or caudaldiscectomy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a summary of various embodiments of methods for generatingtissue-engineered composite intervertebral discs with aligned collagenin the annulus fibrosus according to the present invention. The desireddimensions for a tissue-engineered IVD are obtained by imaging a spinewith CT/MRI. Either the NP structure or the AF structure are firstcreated. The NP structure can be created via injection molding or bycreating a sheet of alginate and then cutting out NP structures of aproper shape. When the NP structure is first created, the NP structureis allowed to crosslink and then a collagen gel is contracted aroundalginate NP during culture to create an AF structure surrounding the NPstructure, thereby forming a completed disc. If the AF structure iscreated first, a mandrel is used and a cell-seeded gel is contractedaround the mandrel during culture, after which cell-seeded alginate isinjected into the lacuna of the contracted AF ring to form the NP. Thestructure is then crosslinked to form the completed disc.

FIG. 2 shows an overview of gel culture and imaging techniques pursuantto various methods of fabricating a tissue-engineered IVD according tothe present invention. Disc and annulus were contracted over 3 daysbefore being segmented for imaging. Images were obtained from the outer(o), middle (m), and inner (i) regions of each gel. Coordinates weredefined in reference to the imaged gel segments.

FIGS. 3A-C illustrate collagen alignment in IVDs of the presentinvention. FIGS. 3A and 3B are images that show the collagen alignmentquantification. FIG. 3A is a second harmonic generation (“SHG”)microscopy image. FIG. 3B is a Fourier transform of the image (contrastadjusted). Fourier amplitude components (fast Fourier transform (“FFT”)image intensities) were summed up along angles at 5° increments from 0to 180° and are represented as an arrow and θ. FIG. 3C is a histogramshowing the summation of amplitudes of the image intensities along each5° increment. From this histogram the mode was calculated, and thealignment index (“AI”) was calculated according to Equation 1 (infra).

FIG. 4 is a graph showing contraction of cell-seeded gels represented asa percentage of the constructs' original surface area. Data arepresented as means±standard deviations for n=7 (*=p<0.05).

FIGS. 5A-5C are SHG-two-photon excited fluorescence (“TPEF”) images fromthe inside region (FIG. 5A) during contraction of 1 and 2.5 mg/mLcollagen disc constructs over 3 days, and magnified image (FIG. 5B)showing aligned fibers between cells on day 3 of contraction in 2.5mg/mL collagen discs. FIG. 5C shows SHG-TPEF images from the insideregion during contraction of 1 and 2.5 mg/mL collagen annular constructsover 3 days.

FIGS. 6A-6D are graphs presenting SHG alignment data. The data is shownfor collagen annular fibrosus gels with AI broken down by day and gelconcentration (n=21) (FIG. 6A); AI further broken down by region of gel(n=7) (O, outside; M, middle; I, inside) (FIG. 6B); and mode anglebroken down by day, concentration, and region of gel for 1 mg/mL (n=4)(FIG. 6C); and 2.5 mg/mL gels (n=7) (FIG. 6D). Data are presented asmeans and standard deviations (#=p<0.05 compared with day 0; *=p<0.05for indicated groups).

FIG. 7 provides photographs showing hematoxylin and eosin staining andTPEF cellular imaging of 1 mg/mL disc gels and annular gels at day 0 and3 of contraction from the inside region of the gel.

FIGS. 8A-8C are photographs showing composite tissue-engineered IVDs(FIGS. 8A-B), and a graph presenting corresponding SHG alignment data(FIG. 8C). FIG. 8A shows a composite disc before contraction withalginate NP in the center of the well and collagen solution pouredaround the alginate NP. FIG. 8B shows a composite disc after 2 weeks ofculture with collagen gel contracted around alginate NP forming atissue-engineered composite intervertebral disc according to oneembodiment of the present invention. The graph of FIG. 8C shows SHGalignment data measured across the entirety of the contracted collagengel thickness, indicating a high degree of collagen alignment in thecircumferential direction at day 14.

FIGS. 9A-B provide structural data (FIG. 9A) and photographs oftissue-engineered IVDs according to one embodiment of the presentinvention (FIG. 9B). FIG. 9A is a pair of graphs showing the equilibriumcompressive modulus (upper graph) and hydraulic permeability (lowergraph) of a tissue-engineered IVD according to the present invention.FIG. 9B is a pair of images showing composite tissue-engineered IVDsproduced by contracting AF-seeded collagen around an NP seeded core(left photograph). The photograph on the right illustrates amulti-lamellar construct produced by successive contraction of collagenlayers, according to one embodiment of the present invention.

FIG. 10 presents SHG and TPEF images of AF seeded collagen gels. Withtime, AF cells elongated and collagen aligned parallel to cells.

FIG. 11 is a graph illustrating an alignment index of AF-seeded collagengels showing time- and concentration-dependent organization.

FIG. 12 is a graph showing the L5-L6 disc height in animals implantedwith a tissue-engineered IVD according to one embodiment of the presentinvention.

FIGS. 13A-D are x-rays of rat vertebrae immediately post-operative (FIG.13A), 1 week (FIG. 13B), 1 month (FIG. 13C), and 3 months (FIG. 13D)after receiving a tissue-engineered IVD of the present invention atL5/L6 (arrow). Note maintenance of disc height and absence of deformity.

FIG. 14 is a photograph showing the appearance of native caudal IVD(left) and a tissue-engineered IVD according to one embodiment of thepresent invention (right).

FIG. 15 is a graph showing CA3-CA4 disc height in controls (Disectomy),animals implanted with a tissue-engineered IVD of the present invention(TE IVD), or their own disc (Native IVD).

FIGS. 16A-D are magnetic resonance images of rat vertebrae: Native disc(FIG. 16A), immediately post-operatively after implantation of TEcomposite (FIG. 16B), 1 month (FIG. 16C), and 6 months after (FIG. 16D).Note hydration (white), maintenance of disc height, and absence ofdeformity.

FIG. 17 presents stained images of tissue-engineered IVDs of the presentinvention at 6 weeks and 6 months in vivo: Picrosirius red (top) andAlcian blue (bottom).

FIG. 18 is a series of photographs illustrating one embodiment of theprocess of fabricating a composite tissue-engineered IVD of the presentinvention. According to the illustrated embodiment, an image-based modelis used to produce an injectable mold. Cell-seeded alginate NP (3% w/v)was created and placed in the center of a 24 well plate and cell-seededcollagen gel (1 mg/ml) contracted around the NP for 2 weeks.

FIG. 19 is a graph showing the contraction of a collagen AF region over14 days of culture represented as a percentage of original area (n=6,*=p<0.05).

FIG. 20 is a graph showing hydraulic permeability for implants made withtwo different cell concentrations (1 and 10 million cells/ml) each atthree collagen concentrations (1, 2, and 3.5 mg/ml) calculated at 60%strain (n=6,*=p<0.05)

FIGS. 21A-B are graphs showing mean stress strain curves (FIG. 21A,equilibrium and FIG. 21B, instantaneous) for 1 million cells/ml seededIVDs according to the present invention (n=6, *=p<0.05).

FIGS. 22A-B are bar graphs showing equilibrium (FIG. 22A) andinstantaneous (FIG. 22B) modulus at 60% strain (n=6, *=p<0.05).

FIGS. 23A-D are photographs showing surgical images of a vertebralcolumn exposed (FIG. 23A), with native disc removed (FIG. 23B), with anengineered disc according to one embodiment of the present invention inthe retracted disc space (FIG. 23C), and completed surgery with anengineered disc of the present invention in place (FIG. 23D).

FIG. 24 is a graph showing disc space of a tissue-engineered IVD of thepresent invention represented as a percentage of original disc spaceover 3 months.

FIG. 25 presents photographs showing gross sections (G1-3), Safranin-O(S1-3), picrosirius red (P1-3), and collagen II immunohistochemistry(“IHC”) (C1-3) histology of tissue-engineered IVDs according to thepresent invention. G1-2, S1-2, P1-2, and C1-2 show tissue development inIVDs that maintained disc space. S3, P3, and C3 show fused vertebralbodies at the site of the implanted disc.

FIG. 26 is a schematic illustration showing the creation of animage-based model to produce total disc dimensions from μCT using thevertebral body surface and NP model from a T2 weighted MRI image. Acombination of the MRI and μCT models produced the image-based model.

FIGS. 27A-D are photographs showing the surgical implantation of atissue-engineered total disc replacement (“TE-TDR”) (also referred toherein as a “tissue-engineered IVD”) implant of the present inventioninto the L4/5 disc space. In FIG. 27A, the L4/5 motion segment isexposed. In FIG. 27B, the native disc is removed via scalpel. In FIG.27C, the vertebral bodies are retracted and the TE-TDR is implanted intothe disc space. In FIG. 27D, the vertebral bodies are released,resulting in a successfully implanted TE-TDR specimen.

FIG. 28 is a graph showing comparisons of disc dimensions between thenative IVD, image-based model, and TE-TDR according to one embodiment ofthe present invention (n=5). Measurement planes are indicated on thepicture of the image-based model (the vertical line represents ananterior-posterior plane, the horizontal line represents a lateralplane).

FIGS. 29A-C show X-ray images of disc space immediately followingsurgery (FIG. 29A) and 4 months following implantation (FIG. 29B). Discheight measurements were obtained over 4 months from 5 implanted animals(FIG. 29C).

FIG. 30 shows histology images for three animals. Gross sections (G1-3),Safranin-O (S1-3), and picrosirius red (P1-3) are shown for all threeanimals. Collagen II IHC shown for animal one (C1a) and three (C3) withmagnified vertebral bone implant interface (C1b) are displayed foranimal one, as well. G1-2, S1-2, P1-2, and C1a-b show tissue developmentin discs that maintained disc space. G3, S3, P3, and C3 show fusedvertebral bodies at the site of the implanted disc.

FIGS. 31A-D are images showing Picrosirius staining (FIG. 31A) andpolarized light showing collagen staining and organization in the AFregion of the disc (FIG. 31B). Increased Safranin-O staining in the NPregion of the disc is shown in FIG. 31C. Integration between theimplanted TE-TDR of the present invention and vertebral body bone isshown in FIG. 31D. (Scale bars=1 mm).

DETAILED DESCRIPTION OF THE INVENTION

Intervertebral discs separate the spinal vertebrae from one another andact as natural shock absorbers by cushioning impacts and absorbing thestress and strain transmitted to the spinal column. Intervertebral disctissues are primarily composed of three regions, the end plates, theannulus fibrosus and the nucleus pulposus. The annulus fibrosus is atough collagen-fiber composite that has an outer rim of type I collagenfibers surrounding a less dense fibrocartilage and a transitional zone.These collagen fibers are organized as cylindrical layers. In each layerthe fibers are parallel to one another; however, the fiber orientationbetween layers varies between 30 and 60 degrees. This organizationprovides support during torsional, bending, and compressive stresses onthe spine. The end plates, which are found at the upper and lowersurfaces of the disc, work in conjunction with the annulus fibrosus tocontain the gel-like matrix of the nucleus pulposus within theintervertebral disc. The nucleus pulposus is made up of a soft matrix ofproteoglycans and randomly oriented type II collagen fibers in water.The proteoglycan and water content are greatest at the center of thedisc and decrease toward the disc periphery. Tissues that effectivelymimic these structures can be produced according to the methodsdescribed herein.

One aspect of the present invention relates to a tissue-engineeredintervertebral disc suitable for total disc replacement in a mammal. TheIVD comprises a nucleus pulposus structure comprising a first populationof living cells that secrete a hydrophilic protein and an annulusfibrosis structure surrounding and in contact with the nucleus pulposusstructure, the annulus fibrosis structure comprising a second populationof living cells and type I collagen. The collagen fibrils in the annulusfibrosis structure are circumferentially aligned around the nucleuspulposus region due to cell-mediated contraction in the annulus fibrosisstructure.

The nucleus pulposus structure of the tissue-engineered IVD of thepresent invention includes a first population of living cells. Incertain embodiments, the cells of the nucleus pulposus structure (andthe annulus fibrosus structure described infra) are seeded into ascaffold, gel, or matrix medium or material. For example, the cells maybe present in a gel, such as a hydrogel. The general preparation ofhydrogel-cell compositions is known in the art. See, e.g., U.S. Pat. No.6,773,713 to Bonassar et al., which is hereby incorporated by referencein its entirety. A “hydrogel” is a substance formed when an organicpolymer (natural or synthetic) is set or solidified to create athree-dimensional open-lattice structure that entraps molecules of wateror other solution to form a gel. The solidification can occur, e.g., byaggregation, coagulation, hydrophobic interactions, or cross-linking.Hydrogels can rapidly solidify to keep the cells evenly suspended withina mold (or around or within another solidified gel) until the gelsolidifies. Hydrogels can also be biocompatible, e.g., not toxic tocells suspended in the hydrogel. Any suitable hydrogel or other materialcan be used. Suitable hydrogel examples include, but are not limited to:(1) hydrogels cross-linked by ions, e.g., sodium alginate; (2)temperature dependent hydrogels that solidify or set at bodytemperature, e.g., PLURONICS™; (3) hydrogels set by exposure to eithervisible or ultraviolet light, e.g., polyethylene glycol polylactic acidcopolymers with acrylate end groups; and (4) hydrogels that are set orsolidified upon a change in pH, e.g., TETRONICS™. Examples of materialsthat can be used to form these different hydrogels includepolysaccharides such as alginate, polyphosphazenes, and polyacrylates,which are crosslinked ionically, or block copolymers such as PLURONICS™(also known as POLOXAMERS™), which arepoly(oxyethylene)-poly(oxypropylene) block polymers solidified bychanges in temperature, or TETRONICS' (also known as POLOXAMINES™),which are poly(oxyethylene)-poly(oxypropylene) block polymers ofethylene diamine solidified by changes in pH.

A wide range of scaffold or matrix materials have been used assubstrates for cell culture and may be suitable for forming the NP (andAF) structure of the tissue-engineered IVDs of the present invention. Ingeneral, NP cells have been cultured in hydrogels with alginate (Aguiaret al., “Notochordal Cells Interact With Nucleus Pulposus Cells:Regulation of Proteoglycan Synthesis,” Exp. Cell Res. 246(1):129-37(1999); Baer et al., “Collagen Gene Expression and Mechanical Propertiesof Intervertebral Disc Cell-Alginate Cultures,” J. Orthop. Res.19(1):2-10 (2001); Mizuno et al., “Tissue-Engineered Composites ofAnulus Fibrosus and Nucleus Pulposus for Intervertebral DiscReplacement,” Spine 29(12):1290-7 (2004); Mizuno et al., “Biomechanicaland Biochemical Characterization of Composite Tissue-EngineeredIntervertebral Discs,” Biomaterials 27(3):362-70 (2006), which arehereby incorporated by reference in their entirety) and gelatin (Gruberand Hanley, “Biologic Strategies for the Therapy of Intervertebral DiscDegeneration,” Expert Opin. Biol. Ther. 3(8):1209-14 (2003); Yang etal., “An In-Vitro Study on Regeneration of Human Nucleus Pulposus byUsing Gelatin/Chondroitin-6-Sulfate/Hyaluronan Tri-Copolymer Scaffold,”Artif. Organs 29(10):806-14 (2005), which are hereby incorporated byreference in their entirety). Both materials are well suited tomaintaining NP cell phenotype and enabling proteoglycan synthesis.

In one embodiment of the present invention, the nucleus pulposusstructure has cells in an alginate gel. A suitable alginate gel may haveabout 3% (w/v) alginate. In another embodiment, the alginate gelcomprises about 0.5% to about 10% (w/v) alginate. Alternatively, thecells of the nucleus pulposus structure are contained in a gelatin.

The nucleus pulposus structure of the tissue-engineered IVD of thepresent invention may include type II collagen. Type II collagen servesto provide mechanical support to the tissue and to resist the osmoticswelling pressure exerted by the hydrophilic proteoglycans.

The first population of cells (i.e., cells of the NP structure) may bepresent in the NP structure at a concentration of about 25×10⁶ cells/ml.In another embodiment, the first population of cells is in a range ofconcentration of about 1×10⁶ cells/ml to about 50×10⁶ cells/ml.

The first population of cells may include nucleus pulposus cells.

Nucleus pulposus (and/or annulus fibrosis) cells present in thetissue-engineered IVD of the present invention may be isolated from anysuitable mammalian source organism, including, without limitation,human, simian, orangutan, monkey, chimpanzee, dog, cat, rat, mouse,horse, cow, pig, and the like. The choice of an animal source for IVDcells is non-trivial, as the time at which the cell population of the NPchanges from notochordal to chondrocytic varies with species. Regardlessof species chosen, cells are preferably obtained from skeletally matureanimals (e.g., murine (Gruber et al., “The Sand Rat Model for DiscDegeneration: Radiologic Characterization of Age-Related Changes:Cross-Sectional and Prospective Analyses,” Spine 27:230-4 (2002), whichis hereby incorporated by reference in its entirety), lapine (Sakai etal., “Immortalization of Human Nucleus Pulposus Cells by a RecombinantSV40 Adenovirus Vector: Establishment of a Novel Cell Line for the Studyof Human Nucleus Pulposus Cells,” Spine 29(14):1515-23 (2004), which ishereby incorporated by reference in its entirety), porcine (Baer et al.,“Collagen Gene Expression and Mechanical Properties of IntervertebralDisc Cell-Alginate Cultures,” J. Orthop. Res. 19(1):2-10 (2001), whichis hereby incorporated by reference in its entirety), ovine (Mizuno etal., “Tissue-Engineered Composites of Anulus Fibrosus and NucleusPulposus for Intervertebral Disc Replacement,” Spine 29(12):1290-7(2004); Mizuno et al., “Biomechanical and Biochemical Characterizationof Composite Tissue-Engineered Intervertebral Discs,” Biomaterials27(3):362-70 (2006), which is hereby incorporated by reference in itsentirety), canine (Aguiar et al., “Notochordal Cells Interact WithNucleus Pulposus Cells: Regulation of Proteoglycan Synthesis,” Exp. CellRes. 246(1):129-37 (1999), which is hereby incorporated by reference inits entirety), bovine (Aguiar et al., “Notochordal Cells Interact WithNucleus Pulposus Cells: Regulation of Proteoglycan Synthesis,” Exp. CellRes. 246(1):129-37 (1999); Alini et al., “The Potential and Limitationsof a Cell-Seeded Collagen/Hyaluronan Scaffold to Engineer anIntervertebral Disc-Like Matrix,” Spine 28(5):446-54 (2003); Seguin etal., “Tissue Engineered Nucleus Pulposus Tissue Formed on a PorousCalcium Polyphosphate Substrate,” Spine 29(12):1299-1306 (2004), whichare hereby incorporated by reference in their entirety), simian, andhuman (Yang et al., “An In-Vitro Study on Regeneration of Human NucleusPulposus by Using Gelatin/Chondroitin-6-Sulfate/Hyaluronan Tri-CopolymerScaffold,” Artif. Organs 29(10):806-14 (2005), which is herebyincorporated by reference in its entirety)).

Suitable cells for the tissue-engineered IVD of the present inventionmay be obtained and/or isolated from essentially any intervertebral disctissue, such as nucleus pulposus or annulus fibrosus tissues. In oneembodiment, cells are obtained from only one type of intervertebral discsource and are not mixed with intervertebral disc cells of another type,e.g., obtained nucleus pulposus cells are essentially free of annulusfibrosus cells. Alternatively, cells can be isolated from bone marrow,adipose, blood, or any other source of mesenchymal stem cells. See, forexample, U.S. Pat. Nos. 5,197,985 and 4,642,120, and Wakitani et al.,“Mesenchymal Cell-Based Repair of Large, Full-Thickness Defects ofArticular Cartilage,” J. Bone Joint Surg. Am. 76:579-591 (1994), whichare hereby incorporated by reference in their entirety.

Intervertebral disc cells can be isolated by any suitable method.Various starting materials and methods for cell isolation are known (seegenerally, Ian Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASICTECHNIQUES (5th ed., 1987); Klagsburn, “Large Scale Preparation ofChondrocytes,” Methods Enzymol. 58:560-564 (1979); Shinmei et al., “TheRole of Interleukin-1 on Proteoglycan Metabolism of Rabbit AnnulusFibrosus Cells Cultured In Vitro,” Spine 13(11): 1284-90 (1988);Maldonado et al., “Initial Characterization of the Metabolism ofIntervertebral Disc Cells Encapsulated in Microspheres,” J. Orthop. Res.10(5): 677-90 (1992), which are hereby incorporated by reference intheir entirety).

Cells can be obtained directly by conventional enzymatic digestion andtissue culture methods, which are described in the Examples (infra).Alternatively, one known method for cell isolation includes differentialadhesion to plastic tissue culture vessels. In another method,antibodies that bind to intervertebral disc cell surface markers can becoated on tissue culture plates and then used selectively to bindintervertebral disc cells from a heterogeneous cell population. In yetanother method, fluorescence activated cell sorting (FACS) usingintervertebral disc-specific antibodies can be used to isolate cells. Instill another method, cells can be isolated on the basis of theirbuoyant density, by centrifugation through a density gradient such asFicoll. These and other methods are well-known in the art.

It may be desirable in certain circumstances to utilize intervertebraldisc stem cells rather than differentiated intervertebral disc cells.Examples of tissues from which stem cells for differentiation, ordifferentiated cells suitable for transdifferentiation, can be isolatedinclude placenta, umbilical cord, bone marrow, blood, fat, skin, muscle,periosteum, and perichondrium. Cells can be isolated from these tissuesthrough an explant culture and/or enzymatic digestion of surroundingmatrix using conventional methods.

In one embodiment of the present invention, cells in the firstpopulation of cells secrete the hydrophilic protein proteoglycan.Particular proteins typically found in the extracellular matrix producedby the cells of the NP may also be secreted by cells in the firstpopulation of cells. Other hydrophilic proteins may also be secreted inaddition to, or alternatively to, proteoglycan. These proteins arecharacterized by their ability to bind water molecules to providecompressible properties to the nucleus pulposus. Suitable hydrophilicproteins may include one or more of chondroitin sulfate, heparansulfate, keratan sulfate, and hyaluronic acid.

Many cartilaginous tissues such as IVDs display a heterogeneous collagenmicrostructure that results in mechanical anisotropy, which isresponsible for mechanical function of the tissue to regulate cellularinteractions and metabolic responses of cells embedded within thesetissues. Using collagen gels seeded with annulus fibrosus cells,constructs of varying structure and heterogeneity may be created tomimic the circumferential alignment of a native IVD. In certainembodiments of the present invention, circumferential alignment may beinduced within gels by contracting annular gels around an inner boundaryusing either, e.g., a polyethylene center or an alginate center tocreate a composite engineered IVD. This alignment can also be producedaccording to certain embodiments of the invention in acomposite-engineered IVD with, e.g., an alginate nucleus pulposus.

Cells of the first population (and/or any other cells used in thepresent invention) may be engineered to express a suitable protein, suchas a hydrophilic protein. For example, nucleic acid molecules encoding ahydrophilic (or any other) protein can be incorporated into host cellsusing conventional recombinant DNA technology. Generally, this involvesinserting the DNA molecule into an expression system to which the DNAmolecule is heterologous (i.e., not normally present). The heterologousDNA molecule is inserted into the expression system or vector in senseorientation and correct reading frame. The vector contains the necessaryelements (promoters, suppressers, operators, transcription terminationsequences, etc.) for the transcription and translation of the insertedprotein-coding sequences. A recombinant gene or DNA construct can beprepared prior to its insertion into an expression vector. For example,using conventional recombinant DNA techniques, a promoter-effective DNAmolecule can be operably coupled 5′ of a DNA molecule encoding theprotein and a transcription termination (i.e., polyadenylation sequence)can be operably coupled 3′ thereof.

Polynucleotides encoding a desired protein can be inserted into anexpression system or vector to which the molecule is heterologous. Theheterologous nucleic acid molecule is inserted into the expressionsystem or vector in proper sense (5′->3′) orientation relative to thepromoter and any other 5′ regulatory molecules, and correct readingframe. The preparation of the nucleic acid constructs can be carried outusing standard cloning methods well known in the art as described bySAMBROOK & RUSSELL, MOLECULAR CLONING: A LABORATORY MANUAL (Cold SpringsLaboratory Press, 2001), which is hereby incorporated by reference inits entirety. U.S. Pat. No. 4,237,224 to Cohen and Boyer, which ishereby incorporated by reference in its entirety, also describes theproduction of expression systems in the form of recombinant plasmidsusing restriction enzyme cleavage and ligation with DNA ligase.

Suitable expression vectors include those which contain replicon andcontrol sequences that are derived from species compatible with the hostcell. For example, if E. coli is used as a host cell, plasmids such aspUC19, pUC18, or pBR322 may be used. When using insect host cells,appropriate transfer vectors compatible with insect host cells include,pVL1392, pVL1393, pAcGP67, and pAcSecG2T, which incorporate a secretorysignal fused to the desired protein, and pAcGHLT and pAcHLT, whichcontain GST and 6×His tags (BD Biosciences, Franklin Lakes, N.J.).Suitable viral vectors include, adenoviral vectors, adeno-associatedviral vectors, vaccinia viral vectors, nodaviral vectors, and retroviralvectors. Other suitable expression vectors are described in SAMBROOK ANDRUSSELL, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs LaboratoryPress, 2001), which is hereby incorporated by reference in its entirety.Many known techniques and protocols for manipulation of nucleic acids,for example in preparation of nucleic acid constructs, mutagenesis,sequencing, introduction of DNA into cells and gene expression, andanalysis of proteins, are described in detail in CURRENT PROTOCOLS INMOLECULAR BIOLOGY (Fred M. Ausubel et al. eds., 2003), which is herebyincorporated by reference in its entirety.

Different genetic signals and processing events control many levels ofgene expression (e.g., DNA transcription and messenger RNA (“mRNA”)translation) and subsequently the amount of desired protein that isproduced and expressed by the host cell. Transcription of DNA isdependent upon the presence of a promoter, which is a DNA sequence thatdirects the binding of RNA polymerase, and thereby promotes mRNAsynthesis. Promoters vary in their “strength” (i.e., their ability topromote transcription). For the purposes of expressing a cloned gene, itis desirable to use strong promoters to obtain a high level oftranscription and, hence, expression. Depending upon the host systemutilized, any one of a number of suitable promoters may be used. Commonpromoters suitable for directing expression in mammalian cells include,without limitation, SV40, MMTV, metallothionein-1, adenovirus Ela, CMV,immediate early, immunoglobulin heavy chain promoter and enhancer, andRSV-LTR. The promoters can be constitutive or, alternatively,tissue-specific or inducible.

The nucleus pulposus structure of the tissue-engineered IVD of thepresent invention may have an isotropic structure.

In addition to its nucleus pulposus structure, the tissue-engineered IVDof the present invention also includes an annulus fibrosus structurethat surrounds the nucleus pulposus structure. Like the nucleus pulposusstructure, the annulus fibrosus structure includes a population ofliving cells, preferably seeded into a gel, matrix, or scaffold, toprovide a medium for structure and cell maintenance and growth. Theannulus fibrosus structure also contains type I collagen. Collagenfibrils in the annulus fibrosus structure are circumferentially alignedaround the nucleus pulposus region as a result of cell-mediatedcontraction in the annulus fibrosus structure.

Many materials have been used for AF cell culture, and are suitable foruse in the tissue-engineered IVD of the present invention. Thesematerials include, without limitation, collagen (both types I and II)(Alini et al., “The Potential and Limitations of a Cell-SeededCollagen/Hyaluronan Scaffold to Engineer an Intervertebral Disc-LikeMatrix,” Spine 28(5):446-54 (2003); Gruber and Hanley, “BiologicStrategies for the Therapy of Intervertebral Disc Degeneration,” ExpertOpin. Biol. Ther. 3(8):1209-14 (2003); Saad and Spector, “Effects ofCollagen Type on the Behavior of Adult Canine Annulus Fibrosus Cells inCollagen-Glycosaminoglycan Scaffolds,” J. Biomed. Mater. Res. A71(2):233-41 (2004), which are hereby incorporated by reference in theirentirety), PGA (Mizuno et al., “Tissue-Engineered Composites of AnulusFibrosus and Nucleus Pulposus for Intervertebral Disc Replacement,”Spine 29(12):1290-7 (2004); Mizuno et al., “Biomechanical andBiochemical Characterization of Composite Tissue-EngineeredIntervertebral Discs,” Biomaterials 27(3):362-70 (2006), which arehereby incorporated by reference in their entirety), small intestinesubmucosa (Le Visage et al. “Small Intestinal Submucosa as a PotentialBioscaffold for Intervertebral Disc Regeneration,” Spine 31(21):2423-30(2006), which is hereby incorporated by reference in its entirety), andchitosan (Dang et al., “Temperature-responsive Hydroxybutyl Chitosan forthe Culture of Mesenchymal Stem Cells and Intervertebral Disk Cells,”Biomaterials 27(3):406-18 (2006), which is hereby incorporated byreference in its entirety).

The second population of cells (i.e., cells of the AF structure) may bepresent in the AF structure at a concentration of about 0.1-5.0×10⁷cells/ml. In another embodiment the second population of cells is in arange of concentration of about 0.1-5.0×10⁶ cells/ml. In anotherembodiment, the second population of cells is at concentration of about1×10⁶ cells/ml, 2×10⁶ cells/ml, 3×10⁶ cells/ml, 4×10⁶ cells/ml, or 5×10⁶cells/ml. The second population of cells may include annulus fibrosuscells.

The annulus fibrosus structure may contain type I collagen at aconcentration of about 1 to about 5 mg/ml, at a concentration of about2.5 to about 5 mg/ml, at a concentration of about 1 to about 30 mg/ml,or at a concentration of about 2.5 to about 30 mg/ml. In a furtherembodiment, the annulus fibrosus structure comprises type I collagen ata concentration of about 1 mg/ml, about 2 mg/ml, about 2.25 mg/ml, about2.5 mg/ml, about 3 mg/ml, about 3.5 mg/ml, about 4 mg/ml, about 4.5mg/ml, or about 5 mg/ml.

The annulus fibrosus structure of the tissue-engineered IVD of thepresent invention may have an anisotropic structure. Thus, in oneembodiment of the present invention, the nucleus pulposus structure hasan isotropic structure and the annulus fibrosis structure has ananisotropic structure.

The nucleus pulposus and/or annulus fibrosus structures of thetissue-engineered IVD of the present invention may be permeable to allownutrient transport to developing tissue. In one embodiment, thecomposite disc has a hydraulic permeability ranging from about 1×10⁻¹¹m²/Pa s to about 3×10⁻¹⁰ m²/Pa s. In other embodiments, the hydraulicpermeability may range from about 1×10⁻¹⁴ m²/Pa s to about 1×10⁻⁹ m²/Pas.

The composite tissue-engineered intervertebral disc of the presentinvention possesses a unique set of mechanical properties to enable itsproper function. In one embodiment, the composite tissue-engineeredintervertebral discs have an equilibrium modulus of about 1 to about 6kPa, an instantaneous modulus of about 5 to about 40 kPa, and ahydraulic permeability of about 1×10⁻¹¹ m²/Pa s to about 3×10⁻¹⁰ m²/Pas. In other embodiments composite tissue-engineered intervertberal discshave an equilibrium modulus of about 1 to about 500 kPa, aninstantaneous modulus of about 5 to about 2000 kPa, and a hydraulicpermeability of about 1×10⁻¹⁴ m²/Pa s to about 1×10⁻⁹ m²/Pa s.

The tissue-engineered IVD of the present invention is suitable for totaldisc replacement in a mammal. The mammal may be selected from any mammalin need of total IVD replacement. In certain embodiments, the mammal maybe in need of total IVD replacement for treatment of or prevention ofdegenerative disc disease or lower back pain. In other embodiments, themammal is selected for use in the study of total IVD replacement. In oneembodiment, the mammal is selected from a mouse, rat, guinea pig,rabbit, dog, cat, pig, sheep, cow, horse, monkey, or human.

It should be understood that the embodiments discussed with respect tothis aspect of the present invention are also contemplated asembodiments relating to other aspects of the invention.

Another aspect of the present invention is a method of fabricating atissue-engineered intervertebral disc suitable for total discreplacement in a mammal. This method involves providing a first gelcomprising a first population of living cells that secrete a hydrophilicprotein; forming the first gel into a predetermined shape and size;providing a second gel comprising a second population of living cellsand type I collagen; contacting the formed first gel with the second gelat a region that extends circumferentially around the first gel; andstoring the first and second gels under conditions effective for thecollagen in the second gel to align circumferentially around the firstgel by self-assembly of collagen due to cell-mediated gel contraction inthe second gel. The first gel forms a nucleus pulposus structure and thesecond gel forms an annulus fibrosus structure surrounding and incontact with the nucleus pulposus structure, thereby fabricating atissue-engineered IVD suitable for total disc replacement in a mammal.

FIG. 1 shows a summary of various embodiments of methods for fabricatingtissue-engineered composite intervertebral discs with aligned collagenin the annulus fibrosus according to this and other aspects of thepresent invention. The desired dimensions for a tissue-engineered IVDare obtained by imaging a spine with CT/MRI. Either the NP structure orthe AF structure are first created. The NP structure can be created viainjection molding or by creating a sheet of alginate and then cuttingout NP structures of a proper shape. When the NP structure is firstcreated, the NP structure is allowed to crosslink and then a collagengel is contracted around alginate NP during culture to create an AFstructure surrounding the NP structure, thereby forming a completeddisc. If the AF structure is created first, a mandrel is used and acell-seeded gel is contracted around the mandrel during culture, afterwhich cell-seeded alginate is injected into the lacuna of the contractedAF ring to form the NP. The structure is then crosslinked to form thecompleted disc.

In one embodiment of the present invention, formation of the second gelis carried out at a temperature of about 37° C. In another embodiment,gel formation is carried out at temperatures ranging from about 20 toabout 37° C. In yet another embodiment the second gel is stored for aperiod of about 3 to about 28 days.

The formation of the first gel (or matrix or scaffold) into apredetermined shape and size is carried out to form a nucleus pulposusstructure of an adequate shape and size for implantation into amammalian vertebrae. In one embodiment of the present invention, thefirst gel is formed by injection molding techniques, such as thosedisclosed in U.S. Pat. No. 6,773,713 to Bonassar et al., which is herebyincorporated by reference in its entirety. Alternatively, the gel may beformed by forming the gel into a sheet and cutting out, e.g., circularstructures of a suitable size for formation of a nucleus pulposusstructure. The thickness of such a sheet may vary depending upon thedesired shape and size of the tissue-engineered IVD being formed.

To add to the structural integrity of the IVD structure, the gels may becross-linked. This technique is taught in U.S. Pat. No. 6,773,713 toBonassar et al., which is hereby incorporated by reference in itsentirety. For example, alginate is an anionic polysaccharide capable ofreversible gelation in the presence of an effective concentration of adivalent cation. A hydrogel can be produced by cross-linking the anionicsalt of alginic acid, a carbohydrate polymer isolated from seaweed, withions, such as calcium cations. The strength of the hydrogel increaseswith either increasing concentrations of calcium ions or alginate. U.S.Pat. No. 4,352,883 (which is hereby incorporated by reference in itsentirety) describes the ionic cross-linking of alginate with divalentcations, in water, at room temperature, to form a hydrogel matrix.

In one embodiment of the present invention, the first gel iscross-linked. In a further embodiment of the present invention the firstgel is cross-linked in the presence of CaCl₂.

The shape and size of the gels (or scaffolds or matrices) according tothe present invention can be determined using image-based constructedmolds. Using this technique, the mold may reproduce the correctanatomical shape and size for the particular mammal (or patient) to betreated. MRI and μCT have been used for guiding the design of atissue-engineered meniscus and bone (Ballyns et al., “An Optical Methodfor Evaluation of Geometric Fidelity for Anatomically Shaped TissueEngineered Constructs,” Tissue Eng. Part C Methods (2009); Ballyns etal., “Image-Guided Tissue Engineering of Anatomically Shaped ImplantsVia MRI and Micro-CT Using Injection Molding,” Tissue Eng. Part A14(7):1195-1202 (2008); Cheah et al., “Automatic Algorithm forGenerating Complex Polyhedral Scaffold Structures for TissueEngineering,” Tissue Eng. 10(3-4):595-610 (2004); Sun et al.,“Computer-Aided Tissue Engineering: Application to Biomimetic Modellingand Design of Tissue Scaffolds,” Biotechnol. Appl. Biochem. 39(Pt1):49-58 (2004); Van Cleynenbreugel et al., “Trabecular Bone ScaffoldingUsing a Biomimetic Approach,” J. Mater. Sci. Mater. Med.13(12):1245-1249 (2002), which are hereby incorporated by reference intheir entirety) and can be employed in designing a tissue-engineered IVDaccording to the present invention. In other embodiments, the dimensionsof the IVD may be altered to enhance the function of the tissue. Forexample, the thickness of the tissue-engineered composite IVD may bedeliberately oversized by a factor of about 1 to about 3 to enable moreeffective fixation, i.e., a “press fit.”

CT and μCT can be used to image the vertebral bodies and provide theouter boundary information of the IVD by examining the surface of thevertebral body (Fields et al., “Role of Trabecular Microarchitecture inWhole-Vertebral Body Biomechanical Behavior,” J. Bone Miner. Res.24(9):1523-1530 (2009), which is hereby incorporated by reference in itsentirety), as well as provide information on the thickness of the discspace. In addition, a T2 weighted MRI image can be used to provideinformation on the NP shape and dimensions (Luoma et al., “Disc Heightand Signal Intensity of the Nucleus Pulposus on Magnetic ResonanceImaging as Indicators of Lumbar Disc Degeneration,” Spine 26(6):680-686(2001), which is hereby incorporated by reference in its entirety).Combining these imaging techniques can produce a model of the native IVDthat can be used to create a tissue-engineered IVD of the presentinvention. Such a technique would be applicable in, e.g., the clinicalsetting and could produce tissue-engineered IVDs tailored to theparticular mammalian patient.

Once the IVD is made, it can be directly implanted, or further cultured,e.g., in vitro, to allow the cells to grow within the hydrogelconstruct, e.g., for a period of about 1 to 30 days. In one embodiment,in vitro culturing is carried out for about 3 to about 28 days.

The fabrication methods of the present invention may be carried out byusing a mandrel or mold in place of the NP gel, around which theAF/collagen solution may be placed under conditions effective for thecollagen in the gel to align circumferentially around the centralmandrel by self-assembly of collagen due to collagen-mediated gelcontraction in the AF/collagen solution. The mandrel or mold may thenlater be replaced with a formed NP gel.

Thus, another aspect of the present invention is a method of fabricatinga tissue-engineered intervertebral disc suitable for total discreplacement in a mammal. This method involves providing a first gelcomprising a first population of living cells that secrete a hydrophilicprotein; providing a second gel comprising a second population of livingcells and type I collagen; forming the second gel around a centralmandrel structure; storing the second gel under conditions effective forthe collagen in the second gel to align circumferentially around thecentral mandrel by self-assembly of collagen due to cell-mediated gelcontraction in the second gel; and replacing the central mandrel withthe first gel. The first gel forms a nucleus pulposus structure and thesecond gel forms an annulus fibrosus structure surrounding and incontact with the nucleus pulposus structure, thereby fabricating atissue-engineered IVD suitable for total disc replacement in a mammal.

Implantation of Living Tissue Constructs can be accomplished as taughtin U.S. Pat. No. 6,773,713 to Bonassar et al., which is herebyincorporated by reference in its entirety. Generally, to implant aliving tissue construct, the implantation site of the mammalian patientcan be exposed by surgical resection and the construct implanteddirectly at that site. Alternatively, if the construct is small enough,the implantation site can be viewed with the aid of, e.g., an endoscope,laparoscope, arthroscope, or esophagoscope, all of which can be modifiedto include a mechanical articulation and delivery system for implantingthe tissue construct through a small incision. During implantation, thesite is cleared of bodily fluids including blood, e.g., with a burst ofair or suction. Thus, tissue-engineered IVDs of the present inventioncan be implanted in a mammalian subject.

EXAMPLES

The following examples illustrate various compositions and methods ofthe invention. The examples are intended to illustrate, but in no waylimit, the scope of the invention.

Example 1—Self-Assembly of Aligned Tissue-Engineered Annulus Fibrosusand Intervertebral Disc Composite Via Collagen Gel Contraction

Using collagen gels seeded with ovine annulus fibrosus cells, constructsof varying structure and heterogeneity were created to mimic thecircumferential alignment of the IVD. Alignment was induced within gelsby contracting annular gels around an inner boundary using both apolyethylene center and alginate center to create a composite engineeredIVD. Collagen alignment and heterogeneity were measured using secondharmonic generation microscopy. Decreasing initial collagen density from2.5 mg/mL to 1 mg/mL produced greater contraction of constructs,resulting in gels that were 55% and 6.2% of the original area afterculture, respectively. As a result, more alignment occurred inannular-shaped 1 mg/mL gels compared with 2.5 mg/mL gels (p<0.05). Thisalignment was also produced in a composite-engineered IVD with alginatenucleus pulposus. The resulting collagen alignment could promote furtheraligned collagen development necessary for the creation of amechanically functional tissue-engineered IVD.

Example 2—Cell Preparation

The cell preparation techniques were based on previously describedtechniques (Mizuno et al., “Biomechanical and BiochemicalCharacterization of Composite Tissue-Engineered Intervertebral Discs,”Biomaterials 27(3):362-70 (2006), which is hereby incorporated byreference in its entirety). Sixteen IVDs were dissected out of thelumbar spine region of four adult skeletally mature (14 month old)Finn/Dorset cross male sheep (Cornell University Sheep Program, Ithaca,N.Y.) and washed in phosphate-buffered saline (“PBS”) (Dulbecco's PBS;Gibco BRL, Grand Island, N.Y.). The AF region of the discs was separatedfrom the NP and dissected into small pieces that were digested in 200 mLof 0.3% w/v collagenase type II (Cappel Worthington Biochemicals,Malvern, Pa.) at 37° C. for 9 h. Digested tissue was filtered through a100 mm nylon mesh (BD Biosciences, Bedford, Mass.) and centrifuged at936 g for 7 min. The cells were washed three times in PBS, counted, andseeded at a density of 2500 cells/cm² in culture flasks with Ham's F-12media (Gibco BRL) containing 10% fetal bovine serum (Gemini BioProducts, Sacramento, Calif.), ascorbic acid (25 μg/mL), penicillin (100IU/mL), streptomycin (100 μg/mL), and amphotericin B (250 ng/mL). Cellswere cultured to confluence at 37° C., 5% CO₂ atmosphere, normoxia, pHof 7.2, and 300 mOsm. After culture, cells were removed from T-150flasks with 0.05% trypsin (Gibco). Cell viability and number werecounted with a hemocytometer and trypan blue vital dye. Cells were thendiluted to the appropriate concentrations and seeded in collagen gels.

Example 3—Collagen Solution Preparation and Collagen ConstructFabrication

Collagen type I was obtained from rat tails using established protocols(Pel-Freez Biologicals, Rogers, Ariz.) (Elsdale and Bard, “CollagenSubstrata for Studies on Cell Behavior,” J. Cell Biol. 54:626-37 (1972),which is hereby incorporated by reference in its entirety). Briefly,tendons were dissected from rat tails and transferred to a solution ofdilute acetic acid (0.1%) at a volume of 80 mL/g of tendon at 48° C. for48 h. The solution was centrifuged at 9000 rpm, and the supernatant wastransferred and centrifuged a second time to remove the unsolubilizedcollagen, blood, and muscle tissue. The solution was then subjected tothe bicinchoninic acid assay (Pierce, Rockford, Ill.) to determine thecollagen concentration of the resulting solution. The stock solution wasstored at 48° C. until needed.

Before producing gels, tissue culture plates were incubated with a 2%bovine serum albumin solution at 37° C. for 1 hour to prevent constructadhesion to tissue culture plates upon gelation. The stock collagensolution was mixed with the appropriate volumes of 1N NaOH, 1×PBS, and10×PBS to return the pH to 7.0, maintain 300 mOsm, and produce theappropriate collagen concentrations for the study (Saltzman et al.,“Three-Dimensional Cell Cultures Mimic Tissues,” Ann. N.Y. Acad. Sci.665:259-73 (1992), which is hereby incorporated by reference in itsentirety). This solution was immediately mixed at a 1:1 ratio with thecell/media solution and pipetted into the appropriate tissue cultureplate and allowed to gel for 30 min at 37° C. After the constructs hadgelled, they were floated with 2 mL of the previously described media.

Example 4—Collagen Disc and Annular Constructs

A total of 70 collagen disc constructs were created by pipetting 1 mL ofcollagen-cell solution into a 24-well tissue culture plate and allowingit to gel. Collagen annular constructs were created by pipetting 1 mL ofthe collagen-cell solution into a 12-well tissue culture plate with a1-cm diameter porous polyethylene disc at the center to yield anannular-shaped collagen ring surrounding the polyethylene. The porousdisc was selected to encourage gel to remain around the disc whenfloated with media. Two groups were made for both construct shapes withfinal collagen concentrations of 2.5 mg/mL and 1 mg/mL and a final cellconcentration of 1×10⁶ cells/mL. The discs were floated with 1 mL ofmedia for the disc constructs and 2 mL of media for annular constructsin each well to maintain a similar degree of floating in the wellsduring culture. Seven constructs per group were used for the LIVE/DEADcell viability assay (Invitrogen, Carlsbad, Calif.) immediately afterconstruction. In addition, constructs were cultured for 3 days andallowed to contract freely with 7 constructs per group being harvestedat 0, 1, 2, and 3 days. At each time point, constructs were digitallyphotographed to quantify construct area, and then fixed withphosphate-buffered formalin for 48 h, with sections of each sample beingutilized for SHG microscopy analysis of collagen fibril orientation andhistology.

Example 5—Composite Discs

Alginate hydrogel NP was produced by mixing 3% (w/v) alginate seededwith 25×10⁶ cells/mL with 2% CaSO₄ at 2:1 ratio and injected betweenglass plates to produce a 2-mm-thick alginate sheet. A 1.5 mm biopsypunch was shaped into NP shape dimensions obtained from rat lumbar discsand NP was punched out of the sheet. Alginate NP was subsequently placedat the center of a 24-well plate and 0.405 mL of 2 mg/mL collagensolution was pipetted around the alginate NP to produce a 2-mm-thickcollagen ring surrounding the 2-mm-thick NP. A total of 21 compositediscs were made and 7 constructs were used for the LIVE/DEAD cellviability assay (Invitrogen) immediately after construction. Gels werethen floated with 1 mL of media and allowed to culture for 0 and 2 weekswith seven discs being harvested at each time point and processed in asimilar manner to annular and disc constructs.

Example 6—Imaging, Microscopy, and Histological Analysis

All constructs were imaged with a digital camera (Canon Powershot G5)and quantitatively analyzed for surface area using the Image J software(NIH, Bethesda, Md.) immediately after harvest on 0, 1, 2, and 3 days.

Procedures of simultaneous SHG microscopy of collagen type I fibrils andTPEF microscopy of cells were based on those described previously (Zoumiet al., “Imaging Cells and Extracellular Matrix In Vivo By UsingSecond-Harmonic Generation and Two-Photon Excited Fluorescence,” Proc.Nat'l. Acad. Sci. U.S.A. 99:11014-9 (2002); Zipfel et al., “Live TissueIntrinsic Emission Microscopy Using Multiphoton-Excited NativeFluorescence and Second Harmonic Generation,” Proc. Nat'l. Acad. Sci.U.S.A. 100:7075-80 (2003), which are hereby incorporated by reference intheir entirety). SHG and TPEF images were obtained using a custom-builtmulti-photon microscope with a Ti:Sapphire mode-locked laser providing100 fs pulses at 80 MHz tuned to a wavelength of 780 nm. Images wereacquired using a BioRad (Hercules, Calif.) 1024 laser scanner coupled toan Olympus (Center Valley, Pa.) 1X-70 inverted microscope. Incidentlight was focused on the sample using either a 40× or a 20× objective.Samples were loaded onto the microscope so that fibrils aligned in thecircumferential direction of the constructs were in the 90° directionaccording to a specified coordinate system (FIG. 2 ). Two-photonfluorescence and back-propagating second harmonic signals were collectedand separated by a dichroic filter into two photomultiplier tubes(“PMTs”). One PMT collected the epi-SHG at 360-410 nm produced by thecollagen type I fibrils, and the other PMT collected TPEF signal at420-500 nm produced by the cells (primarily NADH). For both annular anddisc constructs, SHG images were obtained to study the collagen fibrilorientation throughout contraction. Z-series were collected with a20×/0.7 NA water immersion objective to a depth of 80 μm (9 images at 10μm intervals) at the outer, middle, and inner regions of the gels. Theimages were obtained for four samples for each time point and constructtype. Images were also taken at higher resolution with an Olympus40×/1.3 NA oil objective to observe fibril and cellular interactions.

Collagen fibril orientation was calculated from SHG images with a customMATLAB code based on a previously described technique (Ng et al.,“Interstitial Fluid Flow Induces Myofibroblast Differentiation andCollagen Alignment In Vitro,” J. Cell Sci. 118:4731-9 (2005), which ishereby incorporated by reference in its entirety). This technique hasbeen applied to scanning electron microscopy, and histological andconfocal images, and is applied to SHG images here (Chaudhuri et al., “AFourier Domain Directional Filtering Method For Analysis of CollagenAlignment in Ligaments,” IEEE Trans. Biomed. Eng. 34:509-18 (1987);Pourdeyhimi et al., “Measuring Fiber Orientation in Nonwovens: 3.Fourier Transform,” Textile Res. J. 67:143-151 (1997); Nishimura andAnsell, “Fast Fourier Transform and Filtered Image Analyses of FiberOrientation in OSB,” Wood Sci. Technol. 36:287-307 (2002); van Zuijlenet al., “Morphometry of Dermal Collagen Orientation by Fourier Analysisis Superior to Multi-Observer Assessment,” J. Pathol. 198:284-91 (2002),which are hereby incorporated by reference in their entirety). Thealgorithm relies on the fast fourier transform of the SHG images (FIG.3A). The program summed the intensity of the FFT along lines at 58increments from 0 to 180° (FIG. 3B) via the coordinate system described(FIG. 2 ). The angular distribution of summed intensities wascalculated, representing the relative orientation of the fibrils withinthe image (FIG. 3C). From this histogram, the mode was calculated, whichrepresents the angle of maximum alignment, and using Equation 1 analignment index (AI) was calculated.

$\begin{matrix}{{AI} = \frac{\underset{\theta_{m} - {20{^\circ}}}{\int\limits^{\theta_{m} + {20{^\circ}}}}{I{\partial\theta}}}{( {4{0^{{^\circ}}/1}80^{{^\circ}}} ) \times {\underset{0{^\circ}}{\int\limits^{180{^\circ}}}{I{\partial\theta}}}}} & ( {{Equation}1} )\end{matrix}$

AI ranges were from 1 (unaligned) to 4.5 (complete alignment of fibers).Together, the AI provides the degree of alignment observed, whereas themode angle provides the direction of alignment.

One sample at each time point was fixed for 24 h with 10%phosphate-buffered formalin. The specimens were embedded withinparaffin, and serial sections of 5 mm were cut and stained withhematoxylin and eosin for comparison to SHG and TPEF images.

All statistical analysis was performed using three-factor analysis ofvariance and the Bonferroni post hoc test. The AI parameter was testedfor the effect of time in culture (0, 1, 2, and 3 days), region of gel(outside, middle, and inside), and density of gel (1 mg/mL and 2.5mg/mL).

Example 7—Contraction, Fibril Orientation, and Cellular OrientationResults

On the macro scale, the disc and annular constructs followed a similarcontraction profile (FIG. 4 ). The 2.5 mg/mL discs contracted to 75±2.1%of the original area by day 3 compared with a contraction of 55±4.1% ofthe original area for the 2.5 mg/mL annular gels on day 3. The 1 mg/mLdiscs contracted to 15±1.1% of original area, whereas 1 mg/mL annulargels contracted to 6.2±1.4% by day 3. The 1 mg/mL gels contracted veryquickly from day 0 to 1 and approached a steady state, compared with theslower and more steady contraction seen in the 2.5 mg/mL gels over the 3days. Neither the discs nor the annular constructs showed a change inthickness over the 3 days of contraction. In addition, constructs showedno difference (p<0.05) between groups in viability after constructionwith a mean viability of all groups of 92±2%.

On the micro scale, collagen distribution inside the constructs changedmarkedly during the contraction process (FIG. 5A). At day 0, collagenwas distributed uniformly throughout the sample. Over the course of 3days, the distribution became more heterogeneous in the discs, with morecollagen evident in the pericellular region surrounding AF cells. Thiseffect occurred at both concentrations, but was more pronounced in 1.0mg/mL gels. Further, in regions where cells were in tight proximity,collagen fibers were rearranged to form larger bundles on lines betweencells (FIG. 5B). On a larger length scale, this collagen rearrangementresulted in the development of circumferential collagen fibril andcellular alignment within the annular gels (FIG. 5C).

Regarding fibril orientation, collagen discs showed little change infibril alignment over the 3 days of contraction for both the 1 and 2.5mg/mL discs, as indicated by AI values that ranged from 1.2 to 1.3 over3 days. In contrast to the disc constructs, the annular constructsshowed a large degree of fibril alignment in all regions of theconstructs over the 3 days of contraction (FIGS. 6A-D). The AI of the1.0 mg/mL annular construct increased from <1.3 at day 0 to 1.6 at day1, and remained at 1.6 for day 2 and 3. No significant differences werenoticed between regions of the constructs on the same day when the datawere analyzed based on region.

In 2.5 mg/mL gels, AI increased slowly and more steadily over the 3 daysof contraction, changing from 1.3 on day 1 to 1.4 by day 3 (p<0.05compared with day 0). In contrast to the 1 mg/mL annular gels, the 2.5mg/mL gels showed regional heterogeneity, with the middle regions lessaligned compared with the inner and outer regions over days 1, 2, and 3(three-way analysis of variance, p<0.05). The 1 mg/mL gels showedsignificant increases (p<0.05) in fibril alignment compared with the 2.5mg/mL gels at day 1, 2, and 3. With time, mode angles progressed toward90° and distributions became narrower, indicating a direction ofalignment in the circumferential direction (FIGS. 6C and 6D). Thesetrends were present for both 1.0 and 2.5 mg/mL gels, but were morepronounced for 1.0 mg/mL gels. Overall, the data indicate acircumferential alignment of collagen fibrils resulting from annular gelcontraction around a polyethylene core.

Regarding cellular orientation, disc gels showed no global alignment ofcells over the 3 days of contraction despite showing some evidence ofcellular elongation (FIG. 7 ). However, annular gels showed cellularelongation and circumferential alignment of the cells over the 3 days.The cells developed a spindle-shaped morphology elongated between andparallel to the collagen fibrils similar to the morphology and alignmentobserved in the native IVD.

Composite discs formed in size and shape of rat lumbar IVD. Collagen gelAF analogue seeded with AF cells contracted around alginate NP analogseeded with NP cells. Collagen fibrils produced an AI of 1.57±0.06 inthe circumferential direction (FIGS. 8A-C).

Example 8—Results Show Self-Assembly of an Aligned IVD AF Construct fromSeeded Collagen Gels for Use in an Engineered IVD Composite

The broad goal of this work was to develop a method for self-assembly ofan aligned IVD AF construct from seeded collagen gels that can beemployed in an engineered IVD composite. This study focuses onremodeling of collagen gels by AF cells and the creation of annularconstructs with circumferentially aligned fibrils. Previous efforts tomake IVD tissue-engineered constructs have focused mainly on developingthe compressive properties of the tissue with less focus on thedevelopment of an aligned collagen fibril and cellular architecture inthe AF region to provide the necessary tensile and shear properties.Some work has demonstrated the creation of aligned IVD cells inmicrogrooves (Johnson et al., “Topographical Guidance of IntervertebralDisc Cell Growth In Vitro: Towards the Development of Tissue RepairStrategies for the Anulus Fibrosus,” Eur. Spine J. 15 Suppl 3:S389-96(2006), which is hereby incorporated by reference in its entirety) andcreated tissue-engineered scaffolds with aligned nanoscale fiberorientation for use in AF tissue engineering applications (Nerurkar etal., “Mechanics of Oriented Electrospun Nanofibrous Scaffolds forAnnulus Fibrosus Tissue Engineering,” J. Orthop. Res. 25:1018-26 (2007);Nerurkar et al., “ISSLS Prize Winner: Integrating Theoretical andExperimental Methods for Functional Tissue Engineering of the AnnulusFibrosus,” Spine 33:2691-701 (2008), which are hereby incorporated byreference in their entirety). However, to date, none of these methodshave yielded a composite IVD with aligned collagen fibrils and AF cellsaround an engineered NP.

In this study, the cellular and fibril architecture were controlled bythe boundary conditions imposed on contracting collagen gels. This studydemonstrates that over the 3 days of culture, a steady increase ofcircumferential alignment was observed in both the 2.5 and 1 mg/mL gels(FIG. 7 ) with a fixed inner boundary. The increased alignment observedin the 1 mg/mL annular gel compared with the 2.5 mg/mL annular gel islikely due to the increased contraction observed in the 1 mg/mL annulargel (6.2±1.4% of original area) compared with the 2.5 mg/mL annular gel(55±4.1% of original area). The increase in alignment was consistentwith the profile of the contraction curves of the two concentrations ofgels in the annular gels. Further, similar alignment was observed in acomposite construct with a circumferentially aligned collagen AFcontracted around an alginate NP (FIGS. 8A-C). In contrast to theannular gels and composite, minimal alignment was observed in the discgels at 3 days. The ability to create an unaligned disc structure incombination with the aligned annular gels may be useful in studying theeffects of collagen architecture on tissue development in futurestudies. Overall, this technique enables control of the degree andheterogeniety of alignment through the original collagen concentrationsof the gels and boundary conditions.

Similar techniques have been employed in other tissues to create alignedcollagen fibril structures. However, the results of the current studyshow the ability to use these techniques to create an annular constructwith both circumferentially aligned collagen fibrils and aligned AFcells after 3 days of contraction.

Although the main goal of this study was to create collagen alignment inthe AF region, it is likely that the cell alignment and shape may be ofgreat importance. To produce a mechanically functional tissue from acollagen gel, long-term culture is likely needed. Fibroblasts are knownto increase collagen type I expression when maintained in an alignedspindle shape as compared with a randomly oriented structure and isfurther enhanced with the application of a tensile stimulation (Lee etal., “Nanofiber Alignment and Direction of Mechanical Strain Affect theECM Production of Human ACL Fibroblast,” Biomaterials 26:1261-70 (2005),which is hereby incorporated by reference in its entirety). Thespindle-shaped circumferential cellular alignment (FIG. 7 ) may beadvantageous for the future development of the extracellular matrix inlong-term culture, as well as for priming AF cells for mechanicalstimulation.

The use of SHG-TPEF microscopy enabled the simultaneous study ofcollagen architecture and cell morphology. More collagen was observed inthe pericellular region of the cells in the disc constructs over the 3days of contraction and was greater in the 1 mg/mL gels than in the 2.5mg/mL gels. The increased concentration of collagen within thesepericellular regions could result from newly synthesized collagen orfrom pulling of collagen fibrils into the pericellular region by cells.The observed similar profiles of the gel contraction and the developmentof the increased pericellular collagen between the two concentrations ofgels along with the relatively short culture time suggest a contractionmechanism over a collagen production mechanism. The varying collagenarchitecture demonstrates that although tissue-scale variables, such astotal collagen concentration, regulate mechanical properties, it is alsoimportant to characterize the microscale collagen architecture that mayalso yield insight into the process of tissue assembly.

The SHG-TPEF images also showed a cell-fibril-cell interaction. As thegels contracted, fibrils were aligned between adjacent cells in the discand annular gels (FIG. 5 ). The alignment of collagen networks betweentwo cellular islands seeded in collagen gels has been proposed in modeland experimentally observed by Ohsumi et al., “Three-DimensionalSimulation of Anisotropic Cell-Driven Collagen Gel Compaction,” Biomech.Model Mechanobiol. 7:53-62 (2007) (which is hereby incorporated byreference in its entirety), but can be seen here in the SHG imagesoccurring between individual cells. This provides a mechanism for thealignment of fibrils observed within the annular gels. Fibrils firstbecome stretched between the cells; as the cells pull and contractaround the fixed inner core, the strained fibrils between cells will bepredominately oriented in the circumferential direction due to theimposed physical boundary and circumferential tensile stresses. Thiswould not result in aligned fibrils in unbounded discs as no boundarieshave been applied and the cells will contract isotropically. Further,these data demonstrate that cell patterning can be employed in collagengels to further control the resulting collagen architecture ofcontracted collagen gels.

The fabrication methods of the present invention enable generation ofthe dominant circumferential alignment of the collagen fibrils/cells ina composite engineered IVD. Further, the ability to deposit successivelayers of collagen gels may enable the generation of constructs withmultiple lamellae. As a result, contracting collagen gels provide apowerful tool to create the complex structure of the AF.

Example 9—IVD Tissue Engineering

This experiment relates to the development of compositetissue-engineered IVD with both AF and NP regions. In this experiment,the method of which is generally shown in FIG. 1 , IVD were harvestedfrom sheep, and the AF and NP separated for isolation of cells fromthese tissues (Mizuno et al., “Tissue-Engineered Composites of AnulusFibrosus and Nucleus Pulposus for Intervertebral Disc Replacement,”Spine 29(12):1290-7 (2004); Mizuno et al., “Biomechanical andBiochemical Characterization of Composite Tissue-EngineeredIntervertebral Discs,” Biomaterials 27(3):362-70 (2006), which arehereby incorporated by reference in their entirety). The AF portion ofthe IVD was generated from a fibrous PGA mesh shaped either into ananatomic annulus (Mizuno et al., “Tissue-Engineered Composites of AnulusFibrosus and Nucleus Pulposus for Intervertebral Disc Replacement,”Spine 29(12):1290-7 (2004), which is hereby incorporated by reference inits entirety) or a right cylindrical annulus (Mizuno et al.,“Biomechanical and Biochemical Characterization of CompositeTissue-Engineered Intervertebral Discs,” Biomaterials 27(3):362-70(2006), which is hereby incorporated by reference in its entirety). Thisscaffold was seeded with AF cells, and the center of the construct wasfilled with NP cells in 2% alginate. Implants were placed into thedorsum of athymic mice for up to 16 weeks. Anatomically shaped discs andthe cylindrically shaped discs maintained shape for the duration ofimplantation, with changes in tissue composition apparent on grossinspection (Mizuno et al., “Biomechanical and BiochemicalCharacterization of Composite Tissue-Engineered Intervertebral Discs,”Biomaterials 27(3):362-70 (2006), which is hereby incorporated byreference in its entirety).

Histologic analysis of tissue-engineered IVD samples by Safranin 0staining revealed that AF and NP tissue in early stage constructsappeared quite similar (Mizuno et al., “Biomechanical and BiochemicalCharacterization of Composite Tissue-Engineered Intervertebral Discs,”Biomaterials 27(3):362-70 (2006), which is hereby incorporated byreference in its entirety). However, with time, AF tissue began to takeon a fibrous appearance, although there was no global organization tothe fiber structure, particularly compared to native tissue. Incontrast, NP cells were homogenously distributed in the tissue andproduced a homogenous matrix that stained heavily for proteoglycans.Analysis of tissue generated by this technique demonstrated that AF andNP tissue contained extracellular matrix components consistent withnative IVD phenotype. NP tissue was rich in proteoglycan with verylittle collagen, while AF tissue had abundant collagen and moderateamounts of proteoglycan (Mizuno et al., “Biomechanical and BiochemicalCharacterization of Composite Tissue-Engineered Intervertebral Discs,”Biomaterials 27(3):362-70 (2006), which is hereby incorporated byreference in its entirety). Analysis of collagen by Western blotrevealed that the NP portion of the tissue contained predominantly typeII collagen, with a small amount of type I, while the AF portioncontained predominantly type I, with little to no type II (Mizuno etal., “Tissue-Engineered Composites of Anulus Fibrosus and NucleusPulposus for Intervertebral Disc Replacement,” Spine 29(12):1290-7(2004), which is hereby incorporated by reference in its entirety).

The mechanical properties of tissue-engineered IVD was determined usingunconfined compression tests. Stress relaxation data was fit to aporoelastic model of material behavior to calculate the equilibriumcompressive modulus, which gives a measure of tissue stiffness and thehydraulic permeability, which is related to the ease with which fluidmoves through the tissue. Composite tissue-engineered IVD increased 5fold in compressive modulus to ˜50 kPa at 16 weeks as well as decreased4-fold in hydraulic permeability to ˜5×10⁻¹⁴ m²/Pa s (FIG. 9A). At 16weeks, the compressive modulus was ˜30-40% of native intact IVD, (Ho etal., “Spatially Varying Material Properties of the Rat CaudalIntervertebral Disc,” Spine 31(15):E486-93 (2006), which is herebyincorporated by reference in its entirety) and the hydraulicpermeability was ˜10-12 fold higher than native IVD (Yao et al.,“Effects of Swelling Pressure and Hydraulic Permeability on DynamicCompressive Behavior of Lumbar Annulus Fibrosus,” Ann. Biomed. Eng.30(10):1234-41 (2002), which is hereby incorporated by reference in itsentirety). Thus, while there was significant enhancement in mechanicalproperties, the tissue produced by this method is not comparable infunction to native IVD.

As with all materials, the IVD mechanical properties are related tocomposition and structure. The two main extracellular matrix (“ECM”)components of IVD, proteoglycans and collagen, were both found intissue-engineered IVD in quantities that were typically ˜50% of nativetissue, while mechanical properties differed by factors of 5-10. Thisdisparity suggests that the organization of IVD ECM may also play acritical role in determining mechanical function. Collagen in native AFis organized into circumferentially aligned lamellae that serve toresist radial expansion during axial loading. While some organizationwas observed in the AF portion, neither lamellae nor circumferentialalignment were observed.

To control collagen alignment in tissue-engineered IVD, the phenomenonof collagen gel contraction was employed (Bell et al., “Production of aTissue-Like Structure by Contraction of Collagen Lattices by HumanFibroblasts of Different Proliferative Potential In Vitro,” Proc. Nat'l.Acad. Sci. U.S.A. 76(3):1274-8 (1979), which is hereby incorporated byreference in its entirety). It has been demonstrated that cell-basedcontraction of gels involves mechanical and biochemical remodeling ofcollagen, through integrin-mediated traction forces and proteaseactivity (Phillips and Bonassar, “Matrix Metalloproteinase ActivitySynergizes With Alpha2beta1 Integrins to Enhance Collagen Remodeling,”Exp. Cell Res. 310(1):79-87 (2005); Phillips et al., “FibroblastsRegulate Contractile Force Independent of MMP Activity in 3D-Collagen,”Biochem. Biophys. Res. Commun. 312(3):725-32 (2003), which are herebyincorporated by reference in their entirety). Further, crosslinking canbe used to control the mechanical properties of these gels (Roy et al,“Processing of Type I Collagen Gels Using Non-Enzymatic Glycation,” J.Biomed. Mat. Res. Part A 93(3):843-51 (2010), which is herebyincorporated by reference in its entirety) and ECM assembly by seededgels (Roy et al., “Non-Enzymatic Glycation of Chondrocyte-SeededCollagen Gels for Cartilage Tissue Engineering,” J. Orthop. Res.26(11):1434-9 (2008), which is hereby incorporated by reference in itsentirety). It has also been demonstrated that local collagen alignmentis generated in contracting gels near mechanical boundaries (Costa etal., “Creating Alignment and Anisotropy in Engineered Heart Tissue: Roleof Boundary Conditions in a Model Three-Dimensional Culture System,”Tissue Eng. 9:567-77 (2003), which is hereby incorporated by referencein its entirety). This approach was used to make composite implants withcontracted, aligned collagen, and by repeating the contraction proceduremultiple times, multi-lamellar constructs were produced (FIG. 9B).Tissue collagen alignment was measured using two photon and secondharmonic generation (SHG) microscopy (Bowles et al., “Self-Assembly ofAligned Tissue Engineered Annulus Fibrosus and IVD Composite viaCollagen Gel Contraction,” Tissue Eng. Part A. 16(4):1339-1348 (2010),which is hereby incorporated by reference in its entirety), whichenables real time, non-destructive imaging of collagen withouthistologic stains (Zoumi et al., “Imaging Cells and Extracellular Matrixin Vivo by Using Second-Harmonic Generation and Two-Photon ExcitedFluorescence,” Proc. Nat'l. Acad. Sci. U.S.A. 99(17):11014-9 (2002),which is hereby incorporated by reference in its entirety). Over 3 days,images revealed local alignment of collagen fibrils by AF cells (FIG. 10). Collagen alignment was quantified using custom MATLAB code (Ng etal., “Interstitial Fluid Flow Induces Myofibroblast Differentiation andCollagen Alignment In Vitro,” J. Cell Sci. 118:4731-9 (2005), which ishereby incorporated by reference in its entirety), which enabled thecalculation of an alignment index, defined as the fraction of fibrilsaligned within 20° of the mode of the angular intensity distribution.Collagen alignment in gels increased with time in culture (FIG. 11 ) andwas dependent on initial collagen concentration.

Example 10—Implantation of Tissue-Engineered IVD in L5 to L6 Disc Space

To assess the in vivo function of tissue-engineered IVD, samples made bycontracting 1 mg/ml collagen gels with 1×10⁶ cells/ml around NP-seededalginate gels were cultured for 2 weeks and implanted into the L5-L6space in the spine of athymic rats after discectomy. A total of 6 ratsreceived implants, with 1 animal euthanized at 1 week and 5 at 3 months.Results from this pilot study were very encouraging. Serialx-ray/Faxitron demonstrated that 3 of 5 animals maintained disc height(FIG. 12 ), with no signs of deformity (FIGS. 13A-D). The remaining 2animals had collapsed L5-L6 disc space with some focal kyphosis due to avariation in the surgical technique that has since been abandoned.Histology at 1 week showed maintenance of full disc height, new tissueformation in the disc space, and no sign of inflammation or foreign bodyresponse. These results demonstrate that this method provides a workableplatform for testing the in vivo function of tissue-engineered IVD.

Example 11—Implantation of Tissue-Engineered IVD in CA3-CA4 Disc Space

To assess the in vivo function of tissue-engineered IVD, samples made bycontracting 1 mg/ml collagen gels with 1×10⁶ cells/ml around NP-seededalginate gels were cultured for 2 weeks and implanted into the CA3-CA4space in the spine of athymic rats after discectomy. Composite discswere generated via contracting collagen around the injection-molded NPcore to excellent dimensional tolerance (i.e., to within 7% of thedimensions of the native disc) and greatly resembled the native IVD(FIG. 14 ).

Using this strategy for fabrication of implants, a total of 12 ratsreceived implants, with 6 animals euthanized at 1 month and 6 months.Results from this pilot study were very encouraging. Serial MRIdemonstrated that 12 of 12 animals maintained disc height (FIG. 15 ),with no signs of deformity (FIGS. 16A-D), and showed 90% hydration. Onlythe control animals with discectomy had a collapsed CA3-CA4 disc space.Histology at 1 month showed maintenance of 80% disc height, new tissueformation in the disc space, and no sign of inflammation or foreign bodyresponse. These results indicate that a workable platform for testingthe in vivo function of tissue-engineered IVDs has been developed.

Histological analysis of tissue-engineered IVD implants demonstratedsignificant tissue development over the course of 6 months as well asexcellent integration with the neighboring vertebrae (FIG. 17 ). Bothpicosirius red staining (for collagen) and alcian blue staining (forproteoglycans) revealed that no tissue was formed in the disc spaceafter discectomy. In contrast, tissue-engineered IVD showed distinctzonal structure, with the center NP region staining for proteoglycans,while the outer AF region stained heavily for collagen. This effect wasobserved as early as 6 weeks and was even more pronounced at 6 months.The clefts in the tissue stained with Alcian blue at 6 months aresectioning artifacts, and are not present in the picosirius red sectionsor other histological samples.

Example 12—the Role of Annulus Fibrosus Composition on the MechanicalProperties of Tissue-Engineered IVD

Composite-engineered IVD discs were created by contracting collagen gelsaround an alginate NP thus creating circumferentially aligned collagenfibrils in the engineered AF (Nirmalanandhan et al., “Effects of CellSeeding Density and Collagen Concentration on Contraction Kinetics ofMesenchymal Stem Cell-Seeded Collagen Constructs,” Tissue Eng.12(7):1865-72 (2006), which is hereby incorporated by reference in itsentirety) (See FIG. 18 ). Rat lumbar disc dimensions were obtained fromμCT images and direct measurements of the rat lumber IVD. Dimensionswere then used to create an injection mold of the rat NP. 3% (w/v)alginate seeded with ovine nucleus pulposus cells (25×10⁶ cells/ml) wasinjection molded and alginate NP was placed in the center of a 24 wellplate. Collagen type I gel solution made from rat tail tendon was madeat 3 concentrations (1 mg/ml, 2 mg/ml, and 3.5 mg/ml) and was seededwith ovine annulus fibrosus cells at two seeding densities (1 millioncells/ml and 10 million cells/ml) creating six groups. Collagen solutionwas pipetted around the NP and allowed to gel at 37° C. Constructs werefloated with F12 media supplemented with 10% FBS, 100 IU/ml penicillin,100 μg/ml streptomycin, and 25 μg/ml ascorbic acid. Media was changedevery 3 days and constructs cultured for 2 weeks allowing the collagengel region to contract around alginate NP. Pictures were taken of discsat 0, 6, 12, and 14 days to track contraction of AF region around NP.

At 2 weeks, discs were collected and each group underwent mechanicaltesting. Mechanical testing was carried out under unconfined compressionconditions and strained at 5% increments up to 70% strain. Stressrelaxation curves were collected at each strain increment and data wasfit to poroelastic model to yield the equilibrium modulus, instantaneousmodulus, and hydraulic permeability (Mizuno et al., “Biomechanical andBiochemical Characterization of Composite Tissue-EngineeredIntervertebral Discs,” Biomaterials 27(3):362-70 (2006), which is herebyincorporated by reference in its entirety). Two-way ANOVA with Tukeypost-hoc test was performed.

Both seeding density (p<0.05) and collagen concentration (p<0.05)affected the contraction of the AF portion of compositetissue-engineered IVD (FIG. 19 ). AF contraction decreased as collagenconcentration increased. The effect of cell density was greatest at highcollagen concentrations, and was not significant in 1 or 2 mg/ml gels.

The material property most affected by initial construct composition wasthe effective hydraulic permeability, which was 10-fold lower in 2 mg/mlgels than 1 mg/ml gels and 3-fold lower in 3.5 mg/ml gels than in 1mg/ml gels (FIG. 20 ). The equilibrium stress-strain response wassimilar for all gel compositions (FIG. 21A); however, the instantaneousstress strain response was significantly higher for 3.5 and 2 mg/ml gelsthan 1 mg/ml gels at high strains (FIG. 21B). As a result, theequilibrium modulus was not affected by construct composition, while theinstantaneous modulus was 2-3 fold higher in 2 and 3.5 mg/ml gelscompared to 1 mg/ml gels (FIGS. 22A and 22B).

This study demonstrated the effect of initial cell and collagenconcentration on the material properties of composite tissue-engineeredIVD. The stiffest and least permeable constructs resulted fromconstructs with the highest cell and collagen concentrations. Thelargest changes in mechanical behavior were in the instantaneous modulusand the hydraulic permeability, properties closely related to theability of the tissue to pressurize during loading. The fact that theinstantaneous modulus was 5-10 times higher than the equilibrium modulussuggests that significant pressurization takes place in these samples,as in native IVD. While the effective hydraulic permeability of IVDconstructs reported here is significantly higher than native tissue, itis likely that the local permeability of the AF portion of the constructis much lower than that of the composite disc. This is consistent withthe idea that the AF portion of the composite is providing the bulk ofthe resistance in fluid flow in these composites.

The composite mechanical nature of the disc is not observed until 60%strain (FIG. 21B). It is probable that within these discs the highdegree of strain is necessary to pressurize the NP within thesurrounding AF and allow the composite mechanics to be realized. This isimportant for the design of tissue-engineered total disc replacements,as it will be necessary for them to be designed in a manor that allowsthe NP to be pressurized within the AF and not simply placed in thecenter.

Example 13—Image-Based Tissue Engineering of a Total Intervertebral DiscReplacement for Restoration of Function to the Rat Lumbar Spine

It has not been investigated how TE-TDRs will behave in the native discspace. The implanted disc will be subjected to mechanical loading (Satoet al., “In Vivo Intradiscal Pressure Measurement in Healthy Individualsand in Patients With Ongoing Back Problems,” Spine 24(23):2468-2474(1999), which is hereby incorporated by reference in its entirety),limited nutrient supply (Bibby et al., “The Pathophysiology of theIntervertebral Disc,” Joint Bone Spine 68(6):537-542 (2001), which ishereby incorporated by reference in its entirety), and will need tointegrate with the native tissue. As a result, a successful TE-TDR willneed to be sufficiently stiff to withstand loading in the disc space,sufficiently permeable to allow nutrient transport to the developingtissue, and properly sized to fit into the disc space while sittingflush with the vertebral bodies to facilitate integration. However,currently it is unknown what properties are sufficiently stiff andsufficiently permeable for the successful implant of a TE-TDR into thedisc space.

For this reason, the goal of this study was to determine the extent towhich a composite collagen/alginate TE-TDR can maintain function of therat lumbar spine. Specifically, the work produced an image-basedcollagen/alginate TE-TDR tailored to the L4/L5 disc space of athymicrats and studied the performance of those discs in situ for 4 months.The in vivo performance of composite TE-TDRs was assessed via faxitronx-ray to monitor disc height and histology to characterize themorphology of newly formed tissue and integration of the implant withsurrounding tissues.

Magnetic Resonance Imaging data were used in this experiment, and allMRI image data were acquired using a 3.0 Tesla Magnetic ResonanceImaging system (GE Medical Systems, Milwaukee Wis.) equipped with 50mT/m gradients operating at 150 mT/m/ms. Athymic rats were anesthetizedusing 3%/2% isoflurane for induction and maintenance, respectively. Asealed poly(methyl 2-methylpropenoate) box with intake and exhaust portswas used for imaging that also contained a warming gel pack to aid inmaintaining core body temperature. A Hoult-Deslauriers modularradiofrequency resonator was designed in-house consisting of sixinductively coupled, 19 mm diameter resonant loops arranged in acylindrical geometry of length 35 mm with an inductively coupled driveloop placed at one end.

A 2D axial T₂-weighted fast spin echo (“SE”) sequence was used tovisualize the NP region within the disc. Acquisition parameters includeda 90 ms echo time, a 5500 ms repetition time and a 16-length echo trainusing an 8.0 cm×6.4 cm field of view. A 320×256 matrix was reconstructedto 512×512 providing 0.16 mm×0.16 mm×1.0 mm resolution.

Rat spine from L3-S1 was imaged using an MS-8 Micro-CT Scanner (GEHealthcare, London, Ontario, Canada) at an isotropic resolution of 17μm. Scans were calibrated using an air, water, and mineral standardmaterial (SB3, Gammex, RMI).

μCT data were visualized in Microview (GE Healthcare Inc., Princeton,N.J.) and converted to DICOM format. DICOM files were then imported intoslicOmatic v4.3 (TomoVision, Montreal, Canada) where the bony surfacesof the vertebral bodies were manually segmented to obtain the overallshape and dimensions of the L4/L5 IVD (FIG. 26 ). In addition, thespacing between the vertebral bodies was obtained to determine thetarget thickness of the engineered IVD.

MRI data were imported in DICOM format and segmented manually usingslicOmatic v4.3 to create point cloud images of the NP. Point cloudimages were then converted to surface and solid models of the NP inStudio 4.0 (Geomagic Inc., Research Triangle Park, N.C.) (FIG. 26 ). Asa result, the MRI generated NP data and the μCT generated total discmeasurements were combined to provide the target shape and dimensions ofthe IVD and respective AF and NP. In concordance with the collagencontraction method of creating engineered IVD (Bowles et al.,“Self-Assembly of Aligned Tissue Engineered Annulus Fibrosus and IVDComposite via Collagen Gel Contraction,” Tissue Eng. Part A.16(4):1339-1348 (2010), which is hereby incorporated by reference in itsentirety), the shape and dimensions of the NP were used to createinjectable molds of the NP region of the disc (Ballyns et al.,“Image-Guided Tissue Engineering of Anatomically Shaped Implants Via MRIand Micro-CT Using Injection Molding,” Tissue Eng. Part A14(7):1195-1202 (2008), which is hereby incorporated by reference in itsentirety). Total disc dimensions, NP dimensions, and AF dimensions weremeasured on the AP and lateral plane for the native disc, theimage-based model, and the engineered discs.

Isolation and preparation of AF and NP cells were conducted as describedherein (Mizuno et al., “Tissue-Engineered Composites of Anulus Fibrosusand Nucleus Pulposus for Intervertebral Disc Replacement,” Spine29(12):1290-1297 (2004); discussion 1297-1298; Mizuno et al.,“Biomechanical and Biochemical Characterization of CompositeTissue-Engineered Intervertebral Discs,” Biomaterials 27(3):362-370(2006); Bowles et al., “Self-Assembly of Aligned Tissue-engineeredAnnulus Fibrosus and IVD Composite via Collagen Gel Contraction,” TissueEng. (2009), which are hereby incorporated by reference in theirentirety). Four IVDs were removed from the lumbar region of an adultskeletally mature Fin/Dorset cross male sheep (Cornell University SheepProgram, Ithaca, N.Y.) and washed in phosphate buffered saline (PBS)solution (Dulbecco's Phosphate Buffered Saline, Gibco BRL, Grand Island,N.Y.). The AF and NP were subsequently separated by inspection anddissected into small pieces that were digested in 0.3% collagenase typeII (Cappel Worthington Biochemicals, Malvern, Pa.), with the NP digestedfor six hours and the AF for nine hours. Digested tissue was filteredusing a 100 μm nylon mesh (BD Biosciences, Bedford, Mass.) andcentrifuged at 936×g for seven minutes. The cells were seeded at adensity of 2500 cells/cm² in T150 flasks with Ham's F-12 media (GibcoBRL, Grand Island, N.Y.) supplemented with 10% fetal bovine serum(Gemini Bio Products, Sacramento, Calif.), 25 μg/ml ascorbic acid, 100IU/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin B.Cells were cultured to confluence at 37° C., 5% CO₂ atmosphere, andnormoxia. Following culture, cells were removed from T-150 flasks with0.05% trypsin (Gibco).

Example 14—IVD Construction, Implantation, and Analysis

Engineered IVD were constructed as described supra. Briefly, 3% (w/v)alginate seeded with ovine nucleus pulposus cells (25×10⁶ cells/ml) wasinjection molded (FIG. 18 ) using molds derived from MRI and μCT imagesand alginate NP was placed in the center of a 24 well plate. Collagentype I gel solution (1 mg/ml) made from rat-tail tendon (Elsdale andBard, “Collagen Substrata for Studies on Cell Behavior,” J. Cell Biol.54(3):626-637 (1972), which is hereby incorporated by reference in itsentirety), seeded with ovine annulus fibrosus cells at a density of1×10⁶ cells/ml, was pipetted around the NP and allowed to gel at 37° C.using established protocols (Bowles et al., “Self-Assembly of AlignedTissue-engineered Annulus Fibrosus and IVD Composite via Collagen GelContraction,” Tissue Eng. (2009), which is hereby incorporated byreference in its entirety). Constructs were floated with F12 mediasupplemented with 10% FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin,and 25 μg/ml ascorbic acid. Media was changed every 3 days andconstructs were cultured for 2 weeks allowing the collagen gel region tocontract around alginate NP to the dimensions derived from MRI and μCTdata.

After 2 weeks of in vitro culture, composite discs were implanted intothe lumbar spine of athymic rats (n=5) (FIGS. 27A-D). All animalprocedures were performed in accordance with the guidelines of the IACUCof the Hospital for Special Surgery, New York, N.Y. Rats wereanesthetized using ketamine (‘Ketaset’—100 mg/ml) 80-90 mg/kg, andxylazine (‘Rompun’—20 mg/ml) 5 mg/kg, which were mixed together andadministered intraperitoneally. If necessary, anesthesia was prolongedby administration of isoflurane via nose cone. A modified anteriorapproach was used to approach the lower lumbar spine (Rousseau et al.,“Ventral Approach to the Lumbar Spine of the Sprague-Dawley Rat,” LabAnim. 33(6):43-45 (2004), which is hereby incorporated by reference inits entirety). A method was for the first time established to remove thenative disc and to prepare the disc space for implant insertion. Thevertebral column was exposed and the native IVD (L4/L5) removed. Uponremoval, the L4 and L5 vertebral bodies were minimally retracted toallow the insertion of the engineered disc into the disc space (FIG. 28). The disc space was released to press-fit the implant in place andwound closure was performed in layers. An initial dose of 0.01-0.05mg/kg buprenorphine (Buprenex) was administered intraoperatively orimmediately postoperatively prior to anesthetic recovery. Buprenorphinetreatments were performed for two days postoperatively.

Upon implantation, rats were maintained for 4 months with lateral andanterior-posterior x-ray images taken of the implanted disc spaceimmediately prior to surgery, immediately after surgery, and at 1, 4, 8,12, and 16 weeks to monitor disc height. At 4 months, rats weresacrificed and the motion segments explanted.

Spines and bone samples were cleaned of muscle and preserved in 10%phosphate buffered formalin or 4% paraformaldehyde in 0.05 M cacodylatebuffer, pH 7.4. Samples were fixed at room temperature for 2 days usinga rotator or rocker plate to agitate samples during fixation. Followingan overnight running water rinse, samples were decalcified in 10% EDTAin 0.05 M Tris buffer, pH 7.4, until bone was soft and flexible. Anovernight running water rinse was conducted in a VIP tissue processor toparaffin. Embedded samples were sectioned at 5 micron thickness andsubsequently stained with safranin O for proteolycans, picrosirius redfor collagen, and Hemotoxylin and eosin.

Analysis using immunohistochemistry was also conducted. Paraffinsections were dewaxed in xylene and rehydrated to water by a decreasingconcentration of ethyl alcohol baths. The sections were treated with 3%hydrogen peroxide in PBS to reduce endogenous peroxidase activity in thetissues. A protein block was added to reduce the non-specific bindingbetween antibody and tissue components. The antibody for collagen typeII (Santa Cruz Biotechnology, Santa Cruz, Calif.) was used at aconcentration of 250 μg protein per ml of solution. To enhance the typeII collagen localization, the sections were treated with 1%hyaluronidase in PBS, pH 5.5 for 30 minutes at 37° C., prior to addingthe antibody. After an overnight incubation in primary antibody at 4° C.in a humid chamber, the antibody was rinsed off with PBS, and treatedwith a biotinylated antimouse IgG followed by streptavidin reagentsusing the Vectastain ABC Kit (Vector Laboratories, Burlingame, Calif.)according to manufacturers instructions. The final reaction product wasthe brown deposit created by diaminobenzidine and hydrogen peroxide inthe presence of the conjugated peroxidase.

The results show that a model of the native IVD was obtained from μCTand MM imaging and provided the target dimensions for an engineered IVDto be implanted in the L4/L5 disc space. μCT provided the outer boundaryand thickness of the IVD (FIG. 26 ), which measured ananterior-posterior width of 3.23 mm, a lateral width of 3.8 mm, and athickness of 0.99 mm, while MRI data provided the dimensions for the NPregion of the disc (FIG. 28 ), which measured an anterior posteriorwidth of 1.50 mm and a lateral width of 1.93 mm. The combination of theNP (MRI) and total disc (μCT) data allowed for the dimensions of the AFto be determined with a measured AF width of 0.86 mm on the AP plane and0.93 mm on the lateral plane. The dimensions of the image-derived modelwere within 10% of the manually measured dimensions of the native disc(FIG. 28 ). In addition, the engineered constructs differed by less than7% from those of the native disc. Finally, the process had a high degreeof reproducibility with the standard deviations ranging from 78-278 μm(FIG. 28 ). These standard deviations were within 11.8% of the mean inall measurements.

Upon implantation, none of the rats showed any signs of neurologicaldeficit due to the surgery and implantation. Radiographs indicated thedisc space was fully or partially maintained in three of five animals atthe implanted level (FIGS. 29A-C) after 4 months. Two of the discsfailed rapidly with a complete collapse of the disc space occurring by 4weeks with one disc losing disc space constantly over the 4 months to50% of the original disc space. In each of the animals with collapseddisc space the posterior longitudinal ligament had been removed duringsurgery.

Histologically, the 3 samples that maintained disc height producedtissue with composition reminiscent of native IVD (FIG. 30 ), whilethose that resulted in a collapsed disc space produced a fusion betweenthe vertebral bodies. Implanted tissues were generally locatedanteriorly in the disc space with significant staining observed for bothproteoglycans and collagen in the implanted discs at 4 months (FIG. 30 ,S1-2, P1-2). Collagen II was seen distributed throughout the implanteddisc at 4 months by immunohistochemistry. In addition, properlocalization of proteogylcan and collagen was observed in one of themaintained disc spaces (FIGS. 31A and 31C). The NP region containedintense proteoglycan staining compared to the AF region of the implantwhile the AF region showed increased staining for collagen compared tothe NP region. Good integration was observed between the AF and NPregions of the TE-TDR (FIGS. 31A and 31C) and between the TE-TDR andremnant native IVD (FIG. 30 , S1, P1). Furthermore, picrosirius redstaining and polarized light images indicated that the AF regioncontained collagen organization at 4 months (FIGS. 31A and 31B).Finally, in each of the animals that successfully maintained disc space,good integration was observed between the implanted disc and thevertebral bone (FIG. 31D).

Example 15—Clinically Relevant Imaging Data was Effectively Used toDesign Implants to a High Degree of Geometric Accuracy

The aim of this work was to use MRI and μCT to design a natively sizedtissue-engineered IVD and study the performance of those implants in therat lumbar spine. This research provides a method of using clinicallyrelevant imaging data to design implants to a high degree of geometricaccuracy. Initial characterization of these implants showed that theimplanted TE-TDR was capable of maintaining disc height, integratingwith the vertebral bodies, and developing in the native disc space.

Recently, a number of studies have focused on creating compositeengineered IVD implants containing both an AF and NP. A major concern isproducing a construct that is the correct size and shape for thetargeted disc space. One possible solution is to make generalizedmeasurements and have a number of sizes available during implantation tofind the correct fit; however, this may not be practical in tissueengineering due to the cellular cost of producing multiple sizedconstructs. For this reason, this work focused on using clinicallyapplicable imaging modalities to determine the necessary disc dimensionsfor implantation.

Using μCT and MRI, a model of the desired IVD was successfully producedfrom the native rat disc (FIG. 26 ). In addition, when comparingmeasurements obtained from the actual native disc and that obtained fromimaging, the technique was shown to be quite accurate even for therelatively small rat discs (FIGS. 27A-D) with the maximum deviationsbetween measurements being 99 μm. While used here for the rat, thistechnique could be employed clinically to design tissue-engineered IVDsfor larger animals or humans. It should be noted, however, one would notwant to copy a degenerated NP in a tissue-engineered implant. A morelikely clinical scenario would be to obtain the disc space boundariesusing CT and design the NP within those overall boundaries usingestablished values for the NP relative to the disc or obtain them froman adjacent healthy disc.

This study produced IVD composites using the collagen contractionmethod, which resulted in an AF region with circumferentially alignedcollagen fibrils and a cell-seeded alginate NP. By controlling the NPshape and dimensions through injection molding (FIG. 18 ), an accuratealginate NP was created (FIG. 28 ) compared to the native NP andimage-based model; however, the thickness of the gel was purposelyoversized. The degree of accuracy and repeatability is consistent withprevious work when using alginate injection molding to produce tympanicmembrane patches (Hott et al., “Fabrication of Tissue EngineeredTympanic Membrane Patches Using computer-Aided Design and InjectionMolding,” Laryngoscope 114(7):1290-1295 (2004), which is herebyincorporated by reference in its entirety). The oversized implant wasdesigned to be press fit into the disc space and combat slippage of theengineered disc from the disc space before integration. In addition,this allowed for the alginate and collagen material to be flush with thevertebral ends and promote integration between the native and engineeredtissue. Once the NP was created, the collagen gel AF was contractedaround the alginate NP to the AF dimensions provided by the image-basedmodel. Upon completion, this technique resulted in engineered IVDs thatwere similar in dimension to that of the native IVD and image-basedmeasurements (FIG. 28 ).

Upon implantation into the rat L4/L5 disc space, three of five ratsshowed full or partial disc height maintenance at the implanted level(FIG. 29C). The failure of two of the discs to maintain disc height wasseen in animals in which the posterior supporting tissue, including theposterior lateral ligament (“PLL”), had been more aggressively removed.Removal of the supporting tissue provided a possible mechanism for thedestabilization of the spine and collapse of the disc space. Themaintenance of disc height in the remaining animals shows promise forthis type of implant. This indicates that, under the proper conditions,a contracted collagen/alginate implant can function in the basiccapacity of maintaining disc height despite a relatively low stiffnessof these discs when compared to the modulus of a native disc. Currentwork has focused on the replication of the native mechanical propertiesas a key outcome variable in in vitro IVD tissue engineering (Mizuno etal., “Biomechanical and Biochemical Characterization of CompositeTissue-Engineered Intervertebral Discs,” Biomaterials 27(3):362-370(2006); Nerurkar et al., “Nanofibrous Biologic Laminates Replicate theForm and Function of the Annulus Fibrosus,” Nature mater. 8(12):986-992(2009), which are hereby incorporated by reference in their entirety).However, the data presented here indicates that it may not be necessaryfor clinically viable TE-TDR to fully replicate the native mechanicalproperties before implantation. As a result, this may allow for greaterfocus to be placed on other properties of the TE-TDR, such aspermeability, that would encourage nutrient transport and aid tissueformation and integration within the nutrient limited disc space.

In addition to the maintenance of disc height, the successful implantsshowed patterns of proteoglycan and collagen staining that were similarto native IVD (FIG. 30 ). Significant proteoglycan and collagen stainingwas observed in the implanted tissue at 4 months (FIG. 30 , S1-2, P1-2,C1a-b) indicating a cartilaginous tissue being formed by the constructswithin the disc space. Furthermore, in one of the successful constructsthe NP region showed increased proteoglycan staining and lower collagenstaining in comparison to the AF region (FIGS. 31A and 31C). Polarizedlight indicated that the collagen organization in the AF was maintainedafter 4 months. These findings indicate that these constructs are quitecapable of producing cartilaginous tissues in the disc space environmentand promoting IVD like qualities in their development.

For total tissue-engineered discs to succeed in the disc space, thereneeds to be integration of the implant with the vertebral bodies.Without this occurrence it is likely the disc will not remain in thedisc space over time. This study observed very good integration betweenthe implant and vertebral bodies (FIG. 31D) and remnant IVD (FIG. 30 ,S1, P1) at 4 months. This demonstrates that given the proper conditionstotal tissue-engineered discs can integrate with their environment andcreate a mechanically functioning motion segment. Furthermore, not to beoverlooked is the successful integration of the TE-TDR materials withthe remnant native IVD. The integration indicates that these materialscan be used in annular repair following discectomy and allow for newtissue to be developed in the annular defect and prevent reherniationfollowing surgery.

Overall, this work provides both a method of creating anatomicallyshaped IVDs with clinically relevant imaging modalities, as well asproviding the first insight into how engineered discs perform in thenative disc space. Specifically, anatomically shaped engineered IVDswere seen maintaining disc height in a significant portion of cases,developing cartilaginous like tissue in the disc space, and integratingwith the native bone. These results show that TE-TDRs can be developedin a manner that would be clinically applicable.

Example 16—Transplantation of Composite Tissue-Engineered IntervertebralDiscs to Restore Function to the Rat Spine

To investigate the in vivo function of the contracted collagen gelcomposite discs to survive, integrate, and restore disc function to therat spine, the following experiments were performed.

Composite-engineered IVD discs were created by contracting collagen gelsaround an alginate NP thus creating circumferentially aligned collagenfibrils in the engineered AF, as described supra. Rat lumbar discdimensions were obtained from μCT images and direct measurements of therat lumber IVD. Dimensions were then used to create an injection mold ofthe rat NP. 3% (w/v) alginate seeded with ovine nucleus pulposus cells(25×10⁶ cells/ml) was injection molded and alginate NP was placed in thecenter of a 24 well plate. Collagen type I gel solution (1 mg/ml) madefrom rat tail tendon, seeded with ovine annulus fibrosus cells (1×10⁶cells/ml), was pipetted around the NP and allowed to gel at 37° C.Constructs were floated with F12 media supplemented with 10% FBS, 100IU/ml penicillin, 100 μg/ml streptomycin, and 25 μg/ml ascorbic acid.Media was changed every 3 days and constructs cultured for 2 weeksallowing the collagen gel region to contract around alginate NP.

After 2 weeks of in vitro culture, composite discs were implanted intothe lumbar spine of athymic rats (n=5). All animal procedures wereperformed in accordance with the guidelines of the IACUC. Using ananterior approach the vertebral column was exposed and the native IVD(L4/L5) removed. Upon removal, the L4 and L5 vertebral bodies wereminimally retracted to allow the insertion of the engineered disc intothe disc space (FIGS. 23A-D).

Rats were maintained for 3 months with x-ray images taken of theimplanted disc space immediately prior to surgery, immediately aftersurgery and at 1, 4, 8, and 12 weeks to monitor disc height. At 3months, rats were sacrificed and the tissue explanted for grossmorphology inspection, histology, and immunohistochemistry (IHC).

Upon implantation of engineered IVD constructs, all animals returned tonormal activity levels and showed no signs of neurological deficit.Furthermore, engineered discs fully or partially maintained disc spacein 3 of 5 cases (FIG. 24 ). Upon inspection, it was discovered that inthe 3 cases that failed to maintain full disc height, the posteriorlongitudinal ligament had been severed during surgery. Histology showedthe development of disc like tissue in the disc space containing bothproteoglycans and collagen (FIG. 25 ). In addition, engineered tissueshowed good integration with the adjacent bone and remaining native IVDtissue. Discs were pushed towards the anterior side of the disc space.

This study demonstrates the ability of a composite tissue-engineered IVDto survive, integrate, and restore function to the rat spine. Overall,the study showed that engineered IVD tissues can be implanted andmaintained in the IVD disc space (FIG. 25 ), producing a tissue that wasboth IVD like in its ECM composition (S1-2, P1-2, C1) but alsointegrated with the bone (C2) and remaining native IVD tissue (C1).

It was observed that the discs were found toward the anterior region ofthe disc space indicating that the discs may have had difficultyremaining in the disc space (S1-2). Thus, anchoring or containmentstrategies may be used with implanted engineered discs. In addition, thediscovery that the PLL had been severed in all 3 of the failed discshighlights the need for a stable environment to be present for theimplanted tissue to be successful and integrate with the native tissues.Taken as a whole, this work shows that engineered tissues can form IVDlike tissue and integrate successfully with bone in the lumbar discspace.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

What is claimed:
 1. A tissue-engineered intervertebral disc (IVD)comprising: a nucleus pulposus structure comprising a first populationof living cells and an annulus fibrosis structure surrounding and incontact with the nucleus pulposus structure, the annulus fibrosisstructure comprising a second population of living cells and collagen.2. The tissue-engineered IVD according to claim 1, wherein the nucleuspulposus structure comprises an alginate gel.
 3. The tissue-engineeredIVD according to claim 2, wherein the alginate gel comprises about 0.5%to about 10% (w/v) alginate.
 4. The tissue-engineered IVD according toclaim 1, wherein the first population of cells are present in aconcentration of about 1×10⁶ cells/ml to about 50×10⁶ cells/ml.
 5. Thetissue-engineered IVD according to claim 1, wherein the first populationof cells secrete proteoglycan.
 6. The tissue-engineered IVD according toclaim 1, wherein the first population of cells comprise nucleus pulposuscells.
 7. The tissue-engineered IVD according to claim 6, wherein thenucleus pulposus cells are isolated from one or more of the followingsources: ovine, murine, lapine, porcine, canine, bovine, simian, orhuman.
 8. The tissue-engineered IVD according to claim 1, wherein thenucleus pulposus structure further comprises type II collagen.
 9. Thetissue-engineered IVD according to claim 1, wherein the nucleus pulposushas an isotropic structure.
 10. The tissue-engineered IVD according toclaim 1, wherein the second population of cells comprises annulusfibrosus cells.
 11. The tissue-engineered IVD according to claim 10,wherein the annulus fibrosus cells are isolated from one or more of thefollowing sources: ovine, murine, lapine, porcine, canine, bovine,simian, or human.
 12. The tissue-engineered IVD according to claim 1,wherein the second population of cells are present in a concentration ofabout 0.1-5.0×10⁶ cells/ml.
 13. The tissue-engineered IVD according toclaim 1, wherein the annulus fibrosus structure comprises collagen at aconcentration of about 1 mg/ml to about 30 mg/ml.
 14. Thetissue-engineered IVD according to claim 1, wherein the annulus fibrosushas an anisotropic structure.
 15. The tissue-engineered IVD according toclaim 1, wherein the IVD is permeable to allow nutrient transport todeveloping tissue.